( D ) PRR7 distribution across subcellular fractions ( TOT = whole lysate , Syn = synaptosomes , Nuc = nuclei ) isolated from rat hippocampal tissue . Fib ( Fibrillarin ) , SNAP25 PSD95 were used as , presynaptic postsynaptic markers , respectively .

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(D) PRR7 distribution across subcellular fractions (TOT = whole lysate, Syn = synaptosomes, Nuc = nuclei) isolated from rat hippocampal tissue. Fib (Fibrillarin), SNAP25 and PSD95 were used as nuclear, presynaptic and postsynaptic markers, respectively.


( A ) Immunocytochemistry analyses show that glutamate stimulation ( Glut , 100 µM 5 min + 25 min washout ) increased PRR7 in nucleus . Cycloheximide ( 100 µM ) added 30 min prior to glutamate stimulation did not affect this increase ( Cont = 100 ± 12 . 5 % , Glut = 202 . 1 ± 15 . 2 % ; + cycloheximide Cont = 104 . 9 ± 3 . 6 , Glut = 231 . 4 ± 10 . 9 ) ( n = ~ 30 - 50 ) .

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(A) Immunocytochemistry analyses show that glutamate stimulation (Glut, 100 µM 5 min + 25 min washout) increased PRR7 in the nucleus. Cycloheximide (100 µM) added 30 min prior to glutamate stimulation did not affect this increase (Cont=100±12.5%, Glut=202.1±15.2%; +cycloheximide Cont=104.9±3.6, Glut=231.4±10.9) (n=~30-50 cells).


( B ) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons . Epoxomicin ( 50 nM ) added 20 min prior to stimulation did not affect this decrease ( Cont = 100 ± 3 . 6 % , Glut = 32 . 3 ± 1 . 3 % ; + epoxomicin Cont = 99 . 8 ± 4 . 6 , Glut = 42 . 9 ± 1 . 1 ) . ( n = 14 - 17 dendrites , > 1200 for each group ) . Statistical analyses for ( A ) ( B ) were performed using Mann Whitney .

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(B) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont=100±3.6%, Glut=32.3±1.3%; +epoxomicin Cont=99.8±4.6, Glut=42.9±1.1). (n=14-17 dendrites, >1200 puncta for each group). Statistical analyses for (A) and (B) were performed using the Mann Whitney test.


( C ) of PRR7 immunostaining in cultured hippocampal neurons . Treatments ( APV [ 50 µM , APV - ( 2R ) - - 5 - phosphonovaleric acid ] , CNQX [ 40 µM , 6 - cyano - 7 nitroquinoxaline - 2 , 3 - dione ] , TTX [ 1 µM , tetrodotoxin ] , NMDA [ 50 µM , N - Methyl - D - Aspartate ] ) as indicated . Only APV treatment blocked glutamate dependent increase in PRR7 ( 108 . 1 ± 19 % ) . Statistical significance was determined using one - way ANOVA in conjugation with Sidak post - hoc .

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(C) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons. Treatments (APV [50 µM, APV-(2R)-amino-5-phosphonovaleric acid], CNQX [40 µM, 6-cyano-7 nitroquinoxaline-2,3-dione], TTX [1 µM, tetrodotoxin], NMDA [50 µM, N-Methyl-D-Aspartate]) as indicated. Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 (108.1 ± 19%). Statistical significance was determined using one-way ANOVA in conjugation with Sidak post-hoc test.


( D ) PRR7 - GFP accumulates in nucleus of hippocampal neurons upon NMDA treatment . Depicted are representative averaged confocal ( 30x30 µm2 ) from optical sections ( 300 nm step ) of PRR7 - GFP expressing neurons ( DIV17 ) before after bleaching . Prior to stimulation there was substantial PRR7 - GFP in nucleus due to basal neuronal activity ( upper panel ) . This was reduced by incubation overnight with MK801 ( 10 µM ) TTX ( 1 µM ) ( lower panel ) . NMDA ( 50 µM ) stimulation , but not MK - 801 , led to recovery of PRR7 - GFP following bleaching of ROIs at t0 ` ( blue arrow in E ) . ( E ) of changes in GFP intensities were normalized to time point t0 ` corrected for bleaching . No significant differences in PRR7 - GFPFRAP were found in control ( MK801 ) conditions . GFP intensities are displayed via look - up - table ( LUT ) . Data are represented as mean ± ; n = 9 ( NMDA ) n = 5 ( MK801 ) . was done using one - way ANOVA using Bonferroni post - hoc correction .

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(D) PRR7-GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment. Depicted are representative averaged confocal images (30x30 µm2) from optical sections (300 nm step size) of PRR7-GFP expressing neurons (DIV17) before and after bleaching. Prior to stimulation there was substantial PRR7-GFP in the nucleus due to basal neuronal activity (upper panel). This was reduced by incubation overnight with MK801 (10 µM) and TTX (1 µM) (lower panel). NMDA (50 µM) stimulation, but not MK-801, led to recovery of nuclear PRR7-GFP fluorescence following bleaching of nuclear ROIs at t0`(blue arrow in E).(E) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0` and corrected for bleaching. No significant differences in PRR7-GFPFRAP were found in control (MK801) conditions. GFP intensities are displayed via look-up-table (LUT). Data are represented as mean±SEM; n=9 cells (NMDA) and n=5 cells (MK801). Analysis was done using one-way ANOVA using Bonferroni post-hoc correction.


( F ) PRR7 - mEos translocates from distal dendrites to nucleus upon NMDA treatment . ( Left panels ) Confocal maximal projection of hippocampal neurons expressing PRR7 - mEos before after photoconversion of distal dendrites ( ROIs ) through image z - stack from from green ( 488nm ) to red ( 568nm ) using a UV ( 405 nm ) . ( Right panels ) Averaged projection from planes showing accumulation of photoconverted PRR7 - mEos ( 568nm ) in nucleus following NMDA stimulation ( 50 µM - 30min ) . ( G ) Paired of changes in background corrected intensities of PRR7 - mEos at 0 min 30 min following NMDA stimulation or MK801 treatment . ( Average increase ; NMDA = 617 . 8 ± 47 . 7 % , n = 11 , p < 0 . 0005 ; MK801 = 206 . 1 ± 13 . 6 % , n = 5 , n . s . ) . Statistical significance was determined by two - way ANOVA using Sidak ' s correction for multiple comparisons .

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(F) PRR7-mEos translocates from distal dendrites to the nucleus upon NMDA treatment. (Left panels) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7-mEos before and after photoconversion of distal dendrites (ROIs) through the image z-stack from from green (488nm) to red (568nm) using a UV laser (405 nm). (Right panels) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7-mEos (568nm) in the nucleus following NMDA stimulation (50 µM- 30min).(G) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7-mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment. (Average increase; NMDA=617.8±47.7%, n=11 cells, p<0.0005; MK801=206.1±13.6%, n=5 cells, n.s.). Statistical significance was determined by two-way ANOVA using Sidak's correction for multiple comparisons.


( A ) Co - Immunoprecipitations ( Co - IPs ) of PRR7 with GluN1 , PSD95 GluN2B from rat brain lysates . FT = flowthrough , IgG = normal IgGs .

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(A) Co-Immunoprecipitations (Co-IPs) of PRR7 with GluN1, PSD95 and GluN2B from rat brain lysates. FT = flowthrough, IgG = normal IgGs.


( B ) Co - IPs from HEK293 of c - terminal myc - tagged PRR7 with GluN1 in absence of other NMDAR subunits or PSD95 .

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(B) Co-IPs from HEK293 cells of c-terminal myc-tagged PRR7 with GluN1 in the absence of other NMDAR subunits or PSD95.


( C ) Co - IPs from HEK293 transfected with tagged NMDA or AMPA receptor subunits PRR7 ( myc - tagged GluN2B GluA2 flag - tagged PRR7 ) .

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(C) Co-IPs from HEK293 cells transfected with tagged NMDA or AMPA receptor subunits and PRR7 (myc-tagged GluN2B and GluA2 and flag-tagged PRR7).


( D ) ( top ) Schematics of different PRR7 constructs used for co - IP ( FL = full ) . ( bottom ) PRR7 deletion constructs NMDAR subunits were expressed in HEK293 Co - IPs performed as indicated .

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(D) (top) Schematics of the different PRR7 constructs used for co-IP (FL= full length). (bottom) PRR7 deletion constructs and NMDAR subunits were expressed in HEK293 cells and Co-IPs performed as indicated.


( E ) Co - IPs of PRR7 with GluN1 from high - density neuronal cultures following treatment with NMDA ( 100µM , 30 min , representative of n = 4 ) .

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(E) Co-IPs of PRR7 with GluN1 from high-density primary neuronal cultures following treatment with NMDA (100µM, 30 min, representative of n=4).


( A ) Western blots of lysates of neurons transduced with control lentiviruses ( NT ) or lentiviruses to knockdown ( KD ) or overexpress ( OE ) PRR7 . PRR7 knockdown reduced ( 75 . 6 ± 6 . 9 % ) while PRR7 overexpression ( OE ) increased ( 157 . 6 ± 22 . 8 % ) c - Jun compared to controls . No change was detected in c - Fos . One - way ANOVA was used to analyze results with Dunett ' s post hoc ( n = 16 gels for c - Jun 4 gels for c - Fos ) .

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(A) Western blots of lysates of primary neurons transduced with control lentiviruses (NT) or lentiviruses to knockdown (KD) or overexpress (OE) PRR7. PRR7 knockdown reduced (75.6 ± 6.9%) while PRR7 overexpression (OE) increased (157.6 ± 22.8%) c-Jun levels compared to controls. No change was detected in c-Fos levels. One-way ANOVA test was used to analyze results with Dunett's post hoc test (n=16 gels for c-Jun and 4 gels for c-Fos).


( B ) Control or PRR7 transfected HEK293 were treated with cycloheximide for indicated time points c - Jun were measured by Western blotting . PRR7 expression significantly stabilized c - Jun at 120 min ( 110 . 0 ± 7 . 5 % vs control 68 . 7 ± 7 . 9 % ) 240 minutes ( 83 . 6 ± 5 . 2 % vs control 43 . 7 ± 3 . 4 % ) . Values for c - Jun were normalized to their respective 0 min time point ( n = 4 gels for each group performed blind ) . T - between similar time points were used to analyze results .

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(B) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c-Jun protein levels were measured by Western blotting. PRR7 expression significantly stabilized c-Jun levels at 120 min (110.0 ± 7.5% vs control 68.7 ± 7.9%) and 240 minutes (83.6 ± 5.2% vs control 43.7 ± 3.4%). Values for c-Jun were normalized to their respective 0 min time point (n=4 gels for each group performed blind). T-tests between similar time points were used to analyze results.


( C ) PRR7 , FBW7 / or a control ( PSD95 ) were transfected into HEK293 c - Jun ubiquitination was measured by immunoprecipitating c - Jun blotting for ubiquitin . Ubiquitination of c - Jun in presence of FBW7 ( 178 . 3 ± 26 . 9 % ) was significantly reduced in presence of PRR7 ( 103 . 7 ± 20 . 6 % ) but not a control ( PSD95 ) ( n = 5 gels for groups ) .

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(C) PRR7, FBW7 and/or a control protein (PSD95) were transfected into HEK293 cells and c-Jun ubiquitination was measured by immunoprecipitating c-Jun and blotting for ubiquitin. Ubiquitination of c-Jun in the presence of FBW7 (178.3 ± 26.9%) was significantly reduced in the presence of PRR7 (103.7 ± 20.6%) but not a control protein (PSD95) (n=5 gels for all groups).


( D ) Western blots of c - Junubiquitination in hippocampal neuronal cultures . PRR7 knockdown ( KD ) elevated ( 149 ± 10 . 62 % , n = 6 gels ) c - Junubiquitination level in comparison to control neurons ( NT ) . Statistical significance was determined by t - .

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(D) Western blots of c-Junubiquitination in hippocampal primary neuronal cultures. PRR7 knockdown (KD) elevated (149±10.62%, n=6 gels) c-Junubiquitination level in comparison to control neurons (NT). Statistical significance was determined by t-test.


( E ) Western blots showing knockdown ( KD ) or overexpression ( OE ) of PRR7 in hippocampal neurons do not alter of other FBW7 targets mTOR or Myc .

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(E) Western blots showing knockdown (KD) or overexpression (OE) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc.


( A ) Co - IPs of PRR7 with c - Jun from lysates of HEK293 overexpressing PRR7 .

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(A) Co-IPs of PRR7 with c-Jun from lysates of HEK293 cells overexpressing PRR7.


( B ) Pulldown assays in vitro using purified showed a direct interaction between PRR7 c - Jun .

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(B) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c-Jun.


( C ) Co - IPs of PRR7 with FBW7 from lysates of HEK293 overexpressing PRR7 FBW7 .

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(C) Co-IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7.


( D ) Pulldown assays in vitro using purified PRR7 FBW7 .

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(D) Pulldown assays in vitro using purified PRR7 and FBW7.


( E ) Co - IPs of endogenous PRR7 FBW7 from lysates of ratbrain .

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(E) Co-IPs of endogenous PRR7 and FBW7 from lysates of ratbrain.


( F ) Co - IPs of PRR7 with c - Jun FBW7 from lysates of HEK293 overexpressing PRR7 FBW7 . FBW7 expression increased amount of c - Jun pulled down with PRR7 ( n = 4 gels for each panel , representative blots ) . Lower bar graphs - quantitation for Western blots show c - Jun / FBW7 binding is 41 . 4 ± 13 . 9 % greater in presence of PRR7 ( normalized for FBW7 expression ) . Similarly , c - Jun / PRR7 binding is 97 . 4 ± 38 . 1 % greater in presence of FBW7 ( normalized for PRR7 expression ) . Statistical significance was calculated using unpaired t - .

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(F) Co-IPs of PRR7 with c-Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. FBW7 expression increased the amount of c-Jun pulled down with PRR7 (n=4 gels for each panel, representative blots). Lower bar graphs- quantitation for Western blots show c-Jun/FBW7 binding is 41.4±13.9% greater in the presence of PRR7 (normalized for FBW7 expression). Similarly, c-Jun/PRR7 binding is 97.4±38.1% greater in the presence of FBW7 (normalized for PRR7 expression). Statistical significance was calculated using unpaired t-tests.


( A ) Western blots of phospho - c - Jun in neuronal cultures . of phospho - c - Jun represents a of phospho - c - Jun to total c - Jun ratios were normalized to NT . PRR7 knockdown ( KD ) decreased , while PRR7 overexpression ( OE ) increased phospho - c - Jun ( black bars ) . Glutamate stimulation ( gray bars ) increased phospho - c - Jun / c - Jun in NT but not in KD or OE neurons ( n = 7 gels for groups ) . Significance was determined using two - way ANOVA with Sidak post - hoc .

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(A) Western blots of phospho-c-Jun in primary neuronal cultures. Quantification of phospho-c-Jun represents a ratio of phospho-c-Jun to total c-Jun and all ratios were normalized to NT. PRR7 knockdown (KD) decreased, while PRR7 overexpression (OE) increased phospho-c-Jun levels (black bars). Glutamate stimulation (gray bars) increased phospho-c-Jun/c-Jun ratio in NT but not in KD or OE neurons (n=7 gels for all groups). Significance was determined using two-way ANOVA with Sidak post-hoc test.


( B ) AP - 1 driven luciferase luminescence assays . PRR7 expression in HEK293 increased AP - 1 activity ( 190 . 6 ± 13 . 9 % , n = 12 ; RLU = relative units ) . Statistical significance was determined using t - .

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(B) AP-1 driven luciferase luminescence assays. PRR7 expression in HEK293 cells increased AP-1 activity (190.6 ± 13.9%, n=12; RLU= relative light units). Statistical significance was determined using t-tests.


( D ) Quantitation of using 30 minutes of PI in - situ following 48h of WT - PRR7 - GFP or mNLS - PRR7 - GFP overexpression in hippocampal neuronal cultures ( n = 24 per group ) . represent a max projection of five z - stack sections across .

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(D) Quantitation of cell death using 30 minutes of PI in-situ staining following 48h of WT-PRR7-GFP or mNLS-PRR7-GFP overexpression in primary hippocampal neuronal cultures (n=24 cells per group). Images represent a max projection of five z-stack sections across the cell body.


( E ) NMDA treatment ( 25 min , 100 µM followed by 14h chase ) caused 83 . 6 ± 3 . 5 % in control neurons compared to 60 . 3 ± 4 . 6 % in neurons with PRR7 expression knocked down . A c - Jun inhibitory peptide reduced NMDA - mediated to 66 . 2 ± 6 . 0 % in control neurons to 55 . 6 ± 5 . 3 % in neurons knocked down for PRR7 , but this difference was not statistically significant ( n = 5 independent experiments ) . Two - way ANOVA was used in conjugation with Tukey post - hoc . ( Left ) Representative showing transduced neurons ( green - GFP ) PI ( red ) .

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(E) NMDA treatment (25 min, 100 µM followed by 14h chase) caused 83.6±3.5% cell death in control neurons compared to 60.3±4.6% cell death in neurons with PRR7 expression knocked down. A c-Jun inhibitory peptide reduced NMDA-mediated cell death to 66.2±6.0% in control neurons and to 55.6±5.3% in neurons knocked down for PRR7, but this difference was not statistically significant (n=5 independent experiments). Two-way ANOVA was used in conjugation with Tukey post-hoc test. (Left) Representative images showing transduced neurons (green- GFP) and PI staining (red).


( F ) of in response to NMDA stimulation in presence or absence of PRR7 either WT c - Jun or a dominant negative c - Jun4A ( 4A ) . Overexpression of WT c - Jun caused extensive neuronal ( 84 . 8 ± 2 . 4 % ) that was reduced in neurons knocked down for PRR7 ( 43 . 8 ± 9 . 2 ) . Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity ( 50 . 8 ± 7 . 9 % ) but was not additive with PRR7 knockdown ( 42 . 3 ± 8 . 1 % ) ( n = 3 independent experiments ) . Significance was determined using two - way ANOVA in conjugation with Tukey post - hoc . are representative 10X widefield showing transduced neurons ( green - GFP ) PI ( red ) . Data is presented as mean ± . % values represent comparisons to control .

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(F) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c-Jun or a dominant negative c-Jun4A (4A). Overexpression of WT c-Jun caused extensive neuronal cell death (84.8±2.4%) that was reduced in neurons knocked down for PRR7 (43.8±9.2). Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity (50.8±7.9%) but was not additive with PRR7 knockdown (42.3±8.1%) (n=3 independent experiments). Significance was determined using two-way ANOVA in conjugation with Tukey post-hoc test. Images are representative 10X widefield micrographs showing transduced neurons (green- GFP) and PI staining (red). Data is presented as mean±sem. All % values represent comparisons to control.


C - D . ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination ( B ) , whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination ( C ) . complexes were assembled in vitro on ice in presence of dsDNA ( 32 bp ) their in vitro ubiquitination at 30o C was subsequently monitored in a time - course : at indicative time - points , aliquots of reaction were removed analysed by western blotting using FANCD2 V5 antibodies ( Top ) . For each , data - points from three replicate experiments ( three technical replicates ) were used in fitting to a one - phase association model ( Bottom ) .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination (B), whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination (C). Protein complexes were assembled in vitro on ice in the presence of dsDNA (32 bp) and their in vitro ubiquitination at 30o C was subsequently monitored in a time-course: at indicative time-points, aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies (Top). For each protein complex, data-points from three replicate experiments (three technical replicates) were used in fitting to a one-phase association model (Bottom).


A . USP1 - UAF1 - mediated deubiquitination of V5 - FANCI FANCD2 was assessed in absence or presence of ( 51 bp ) , when ubiquitinated versions of these were in isolation ( IUb D2Ub ) or within singly / doubly ubiquitinated ID2 complexes ( IUbD2 , IUbD2Ub ID2Ub ) . At indicated time - points , aliquots of each reaction were removed analysed by western blotting using FANCD2 V5 antibodies .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

A. USP1-UAF1-mediated deubiquitination of V5-FANCI and FANCD2 was assessed in the absence or presence of DNA (51 bp), when ubiquitinated versions of these proteins were in isolation (IUb and D2Ub) or within singly/doubly ubiquitinated ID2 complexes (IUbD2, IUbD2Ub and ID2Ub). At indicated time-points, aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies.


C . Deletion of N - terminus ( ∆ N ) of USP1 ( residues 1 - 54 ) results in greatly reduced FANCD2 deubiquitination . Assays were performed in triplicate ( technical replicates ) as in A , but reactions contained . Left : Western blotting of reaction products at zero 30 minutes using FANCD2 V5 antibodies . Right : Residual FANCI FANCD2 ubiquitination following USP1 - UAF1 treatment for 30 min . Replicate residual ubiquitination values statistically significant changes ( one - way ANOVA with Bonferroni correction ) are shown . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Deletion of N-terminus (∆N) of USP1 (residues 1-54) results in greatly reduced FANCD2 deubiquitination. Assays were performed in triplicate (technical replicates) as in A, but all reactions contained DNA. Left: Western blotting of reaction products at zero and 30 minutes using FANCD2 and V5 antibodies. Right: Residual FANCI and FANCD2 ubiquitination following USP1-UAF1 treatment for 30 min. Replicate residual ubiquitination values and statistically significant changes (one-way ANOVA test with Bonferroni correction) are shown. ***p<0.001.


Alanine mutagenesis of key FANCD2 ' s residues ( H209 , V243 P244 ) participating in extended FANCD2 - ubiquitin interface ( IUbD2 - IUbD2Ub - structures ) , results in enhanced FANCI deubiquitination by USP1 - UAF1 . C . FANCI deubiquitination by USP1 - UAF1 ( incubation with 50 nM USP1 - UAF1 for 30 minutes at room ) in presence of , in absence or presence of wild - type ( D2WT ) or mutant ( D2H209A , VP243AA ) FANCD2 . Replicate residual ubiquitination values ( three technical replicates ) statistically significant changes ( t - one - way ANOVA with Bonferroni correction ) are shown .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Alanine mutagenesis of key FANCD2's residues (H209, V243 and P244) participating in the extended FANCD2-ubiquitin interface (IUbD2-DNA and IUbD2Ub-DNA structures), results in enhanced FANCI deubiquitination by USP1-UAF1. C. FANCI deubiquitination by USP1-UAF1 (incubation with 50 nM USP1-UAF1 for 30 minutes at room temperature) in the presence of DNA and, in the absence or presence of wild-type (D2WT) or mutant (D2H209A, VP243AA) FANCD2. Replicate residual ubiquitination values (three technical replicates) and statistically significant changes (t-test and one-way ANOVA test with Bonferroni correction) are shown.


D . FANCI was ubiquitinated with DyLight - 680 labelled ubiquitin ( IUb - DL680 ) its deubiquitination by USP1 - UAF1 ( incubation with 100 nM USP1 - UAF1 for 20 minutes at room ) , was assessed at increasing concentrations ( 20 , 200 or 200 nM ) of , either wild - type ( D2WT ) , or mutant ( D2H209A , VP243AA ) FANCD2 . Experiment was repeated three times ( technical replicates ) , fold change in residual FANCI ubiquitination was determined by normalisation to mean of IUb - DL680 at 20 nM FANCD2 ( average from six values , for both D2WT D2H209A , VP243AA ) . Mean ( with range ) fold - increase values statistically significant changes upon FANCD2 mutation for each FANCD2 ( t - two - way ANOVA with Bonferroni correction ) are shown .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

D. FANCI was ubiquitinated with DyLight-680 labelled ubiquitin (IUb-DL680) and its deubiquitination by USP1-UAF1 (incubation with 100 nM USP1-UAF1 for 20 minutes at room temperature), was assessed at increasing concentrations (20, 200 or 200 nM) of, either wild-type (D2WT), or mutant (D2H209A, VP243AA) FANCD2. Experiment was repeated three times (technical replicates), and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb-DL680 at 20 nM FANCD2 (average from six values, for both D2WT and D2H209A, VP243AA). Mean (with range) fold-increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration (t-test and two-way ANOVA test with Bonferroni correction) are shown.


( B ) Wild type mice received GFP - K79 via tail vein 1 hr later , were perfused meninges , cortex choroid plexus were obtained for demonstration of intravenously injected . arrows represent co - localized with capillaries ( e . g . , within capillaries ) arrowheads represent outside capillaries ( e . g . , exited from capillaries ) . A few were successfully passed through meningeal cortex capillaries , but no were demonstrated in choroid plexus at this time .

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(B) Wild type mice received strain GFP-K79 via the tail vein and 1 hr later, animals were perfused and the meninges, cortex and choroid plexus were obtained for demonstration of intravenously injected bacteria. The arrows represent bacteria co-localized with capillaries (e.g., within capillaries) and arrowheads represent bacteria outside the capillaries (e.g., exited from the capillaries). A few bacteria were successfully passed through the meningeal and cortex capillaries, but no bacteria were demonstrated in the choroid plexus at this time.


( C ) Sulfo - NHS - biotin was administered via intraperitoneal injection 10 min before perfusion for assessing blood - brain barrier permeability , i . e . , extravasation is indicative of leakage from intravascular lumen . Sulfo - NHS - Biotin was confined to capillaries of meninges cortex ( as shown with arrows ) , there was no evidence of extravasation . Scale bar = 100 μm .

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(C) Sulfo-NHS-biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood-brain barrier permeability, i.e., extravasation is indicative of leakage from the intravascular lumen. Sulfo-NHS-Biotin was confined to the capillaries of the meninges and cortex (as shown with arrows), and there was no evidence of extravasation. Scale bar = 100 μm.


( D ) Relative invasion frequency of GBS K79 in HBMEC with or without EGFR inhibitor ( gefitinib ) .

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(D) Relative invasion frequency of GBS strain K79 in HBMEC with or without EGFR inhibitor (gefitinib).


( E ) recovered from blood brain in wild type mice receiving vehicle control ( n = 5 ) or gefitinib ( 10 mg / kg ) ( n = 6 ) , infected with K79 for 1 h .

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(E) Bacterial counts recovered from the blood and brain in wild type mice receiving vehicle control (n = 5) or gefitinib (10 mg/kg) (n = 6), infected with strain K79 for 1 h.


( F ) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS K79 infection at 15 min 60 min post infection .

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(F) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS strain K79 infection at 15 min and 60 min post infection.


( G ) EGFR expression in EGFR knockout HBMEC using CRISPR / Cas9 ( left panel ) relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout control HBMEC ( right panel ) . From left to right , p value of K79 is 0 . 0059 , p value of K160 is 0 . 00024 , p value of K161 is 0 . 00080 , p value of K181 is 0 . 00042 , p value of K226 is 0 . 00011 , p value of K237 is 0 . 00072 , p value of K238 is 0 . 0093 , p value of P539 is 0 . 00013 ( right panel ) .

O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) EGFR protein expression in EGFR knockout HBMEC using CRISPR/Cas9 (left panel) and relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout and control HBMEC (right panel). From left to right, p value of K79 is 0.0059, p value of K160 is 0.00024, p value of K161 is 0.00080, p value of K181 is 0.00042, p value of K226 is 0.00011, p value of K237 is 0.00072, p value of K238 is 0.0093, p value of P539 is 0.00013 (right panel).


( H ) GBS K79 traversal of HBMEC monolayer was significantly decreased in EGFR knockout HBMEC compare to control HBMEC .

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(H) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in EGFR knockout HBMEC compare to control HBMEC.


( I ) recovered from blood brain of EGFR conditional knockout ( n = 5 ) control mice ( n = 5 ) 1 h after intravenous inoculation with K79 .

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(I) Bacterial counts recovered from the blood and brain of EGFR conditional knockout (n = 5) and control mice (n = 5) 1 h after intravenous inoculation with strain K79.


( A ) Tyrosine phosphorylation of EGFR in HBMEC pre - treated with 20 μM S1P2 antagonist ( JTE - 013 ) or vehicle control infected with GBS K79 infection for 60 min . EGFR phosphorylation in control S1P2 knockout HBMEC infected with K79 .

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(A) Tyrosine phosphorylation of EGFR in HBMEC pre-treated with 20 μM S1P2 antagonist (JTE-013) or vehicle control and infected with GBS strain K79 infection for 60 min. EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79.


( B ) Relative invasion frequency of K79 in HBMEC with or without SphK1 SphK2 inhibitors . inhibitors were used at 10 μM . From left to right , p value of DJB - V - 39 is 0 . 0047 , p value of SKI is 0 . 0062 , p value of FTY720 is 0 . 0022 , p value of HB56 is 0 . 0045 .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors. All the inhibitors were used at 10 μM. From left to right, p value of DJB-V-39 is 0.0047, p value of SKI is 0.0062, p value of FTY720 is 0.0022, and p value of HB56 is 0.0045.


( C ) S1P2 expression in S1P2 knockout HBMEC using CRISPR / Cas9 ( left panel ) . Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC . From left to right , p value of K79 is 0 . 0018 , p value of K160 is 0 . 0091 , p value of K161 is 0 . 0054 , p value of K181 is 0 . 00040 , p value of K226 is 0 . 00032 , p value of K237 is 0 . 040 , p value of K238 is 0 . 041 p value of P539 is 0 . 0025 .

O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR/Cas9 (left panel). Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC. From left to right, p value of K79 is 0.0018, p value of K160 is 0.0091, p value of K161 is 0.0054, p value of K181 is 0.00040, p value of K226 is 0.00032, p value of K237 is 0.040, p value of K238 is 0.041 and p value of P539 is 0.0025.


( D ) GBS K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

(D) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control.


( E ) recovered from blood brain of wild type ( n = 5 ) S1P2 - / - mice ( n = 5 ) infected with K79 for 1h .

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(E) Bacterial counts recovered from the blood and brain of wild type (n = 5) and S1P2-/- mice (n = 5) infected with strain K79 for 1h.


( A ) Serine phosphorylation of cPLA2α in response to GBS K79 in HBMEC pretreated with gefitinib or vehicle control in EGFR knockout HBMEC .

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(A) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC.


( B ) Serine phosphorylation of cPLA2α in response to GBS K79 in control , EGFR knockout S1P2 knockout HBMEC .

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(B) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control, EGFR knockout and S1P2 knockout HBMEC.


( C ) recovered from blood brain in wild type mice ( n = 5 ) CysLT2 - / - mice ( n = 5 ) infected with K79 for 1h .

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(C) Bacterial counts recovered from the blood and brain in wild type mice (n = 5) and CysLT2-/- mice (n = 5) infected with strain K79 for 1h.


( D ) CysLT1 expression in CysLT1 knockdown HBMEC using shRNA ( left panel ) relative invasion frequency of 8 GBS isolates in CysLT1 knockdown control HBMEC . t - . From left to right , p value of K79 is 0 . 0039 , p value of K160 is 0 . 0082 , p value of K161 is 0 . 00091 , p value of K181 is 0 . 0087 , p value of K226 is 0 . 00020 , p value of K237 is 0 . 0039 , p value of K238 is 0 . 00066 , p value of P539 is 1 . 76E - 05 ( right panel ) .

O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA (left panel) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC. t-test. From left to right, p value of K79 is 0.0039, p value of K160 is 0.0082, p value of K161 is 0.00091, p value of K181 is 0.0087, p value of K226 is 0.00020, p value of K237 is 0.0039, p value of K238 is 0.00066, p value of P539 is 1.76E-05 (right panel).


( E ) GBS K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control .

(E) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control.


( F ) recovered from blood brain in wild type mice ( n = 7 ) CysLT1 - / - mice ( n = 7 ) infected with K79 for 1h .

(F) Bacterial counts recovered from the blood and brain in wild type mice (n = 7) and CysLT1-/- mice (n = 7) infected with strain K79 for 1h.


( G ) Ezrin expression in ezrin knockdown HBMEC using shRNA ( left panel ) relative invasion frequency of GBS K79 in ezrin knockdown control HBMEC ( right panel ) .

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(G) Ezrin protein expression in ezrin knockdown HBMEC using shRNA (left panel) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC (right panel).


( H Ezrin phosphorylation in HBMEC pre - treated with CysLT1 antagonist ( montelukast ) infected with GBS K79 for 60 min ( left panel )

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(H Ezrin phosphorylation in HBMEC pre-treated with CysLT1 antagonist (montelukast) and infected with GBS strain K79 for 60 min (left panel)


I ) ezrin phosphorylation in homogenates of brain capillaries of CysLT1 - / - control mice infected with GBS K79 for 1h ( right panel ) .

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I) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1-/- and control mice infected with GBS strain K79 for 1h (right panel).


( J ) Intracellular K79 co - localization with EGFR ezrin in control S1P2 knockout HBMEC ( as shown by arrows , cyan ) . of co - localization was calculated by counting numbers of GBS co - localized intracellular GBS from at least three representative fields , expressing as numbers of co - localized GBS / GBS x 100 .

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(J) Intracellular K79 co-localization with EGFR and ezrin in control and S1P2 knockout HBMEC (as shown by arrows, cyan). Percentage of co-localization was calculated by counting numbers of all GBS and co-localized intracellular GBS from at least three representative fields, and expressing as numbers of co-localized GBS/all GBS x 100.


( A ) Survival of wild type mice receiving drug administration for 7 days after infection with GBS K79 . Data are presented as a Kaplan - Maier plot with a log rank used to compare of survival between groups , CFX group n = 18 , CFX + MONT group n = 10 * p = 0 . 015 . ( CFX : ceftriaxone ; MONT : montelukast ) .

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(A) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79. Data are presented as a Kaplan-Maier plot with a log rank test used to compare percentage of survival between the groups, CFX group n = 18, and CFX + MONT group n = 10*p = 0.015. (CFX: ceftriaxone; MONT: montelukast).


( B ) Nissl of hippocampus CA1 , CA2 & CA3 regions ( low magnification ) cortex region ( higher magnification ) ( left ) histograms showing number of Nissl - stained bodies ( right ) Scale bar , 50 μm . Data represent means ± from three independent experiments , with statistical by One - way ANOVA , p value between control group Ceftriaxone group is 0 . 00021 , p value between Ceftriaxone group Ceftriaxone + Montelukast group is 0 . 013 .

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(B) Nissl staining of the hippocampus CA1, CA2 & CA3 regions (low magnification) and cortex region (higher magnification) (left) and histograms showing the number of Nissl-stained bodies (right) Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value between control group and Ceftriaxone group is 0.00021, p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0.013.


( C - D ) Representative immunofluorescence ( left ) ( right ) of TUNEL bodies , astrocyte ( GFAP ) , microglia ( Iba1 ) in cortex of wild type mice after 7 days of therapy . Scale bar , 50 μm . Data represent means ± from three independent experiments , with statistical by One - way ANOVA , p value of TUNEL bodies between CFX group MONT + CFX group is 0 . 0012 , p value of astrocyte between CFX group MONT + CFX group is 0 . 0068 , p value of microglia between CFX group MONT + CFX group is 0 . 0029 . ( E ) Y - maze for spatial learning memory of different group of wild type mice . total duration of time spent at novel arm ( left ) of entries to novel arm of Y - maze ( right ) . Data represent means ± was statistically analyzed by One - way ANOVA , p value of total duration of time spent at novel arm between control group ( n = 6 ) CFX group ( n = 5 ) is 0 . 00012 , p value between CFX group ( n = 5 ) MONT + CFX group ( n = 6 ) is 0 . 00017 ; p value of total duration of % of entry to novel arm between control group ( n = 6 ) CFX group ( n = 5 ) is 0 . 0102 , p value between CFX group ( n = 5 ) MONT + CFX group ( n = 6 ) is 0 . 0216 .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Representative immunofluorescence images (left) and quantification (right) of TUNEL bodies, astrocyte (GFAP), microglia (Iba1) in cortex of wild type mice after 7 days of therapy. Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value of TUNEL bodies between CFX group and MONT + CFX group is 0.0012, p value of astrocyte between CFX group and MONT + CFX group is 0.0068, and p value of microglia cells between CFX group and MONT + CFX group is 0.0029. (E) Y-maze test for spatial learning and memory of different group of wild type mice. The total duration of time spent at novel arm (left) and the percentage of entries to novel arm of Y-maze (right). Data represent the means ± SEM was statistically analyzed by One-way ANOVA, p value of total duration of time spent at novel arm between control group (n = 6) and CFX group (n = 5) is 0.00012, p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.00017; p value of total duration of % of entry to novel arm between control group (n = 6) and CFX group (n = 5) is 0.0102, and p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.0216.


Wip1 represses gene responses induced by Nodal or BMP signaling in caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in absence of reverse transcriptase . WE , whole . Co AC , uninjected control caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , Xnr1 ( 50 pg )

B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), Xnr1 (50 pg)


Wip1 represses gene responses induced by Nodal or BMP signaling in caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in absence of reverse transcriptase . WE , whole . Co AC , uninjected control caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , BMP4 ( 100 pg ) .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), BMP4 (100 pg).


Wip1 represses gene responses induced by Nodal or BMP signaling in caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in absence of reverse transcriptase . WE , whole . Co AC , uninjected control caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng )

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng)


( D wt Wip1 , but not Wip1 ( D277A ) , down - regulates activity of Activin - or BMP - responsive promoters in HEK293T . FAST1 , were co - transfected as indicated for efficient activation of promoters . Data are expressed as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - .

O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. FAST1, were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


E ) wt Wip1 , but not Wip1 ( D277A ) , down - regulates activity of Activin - or BMP - responsive promoters in HEK293T . Smad1 Smad4 were co - transfected as indicated for efficient activation of promoters . Data are expressed as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - .

O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. Smad1 and Smad4 were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


( F - Q ) Dorsal ( F - N ) or ventral ( O - Q ) injection of wt Wip1 abrogates in vivo expression of mesodermal markers at stage 10 . 25 as analyzed by in situ hybridization . are shown in vegetal views with dorsal to top . Arrowheads denote absence of expression of mesodermal markers in injected region of . Control , an injected with LacZ only . amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) LacZ ( 100 pg ) . Scale bar , 150 μm .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(F-Q) Dorsal (F-N) or ventral (O-Q) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10.25 as analyzed by in situ hybridization. Embryos are shown in vegetal views with dorsal to the top. Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos. Control, an embryo injected with LacZ only. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng) and LacZ (100 pg). Scale bar, 150 μm.


( A , Nodal - induced gene responses are enhanced further in absence of Wip1 as analyzed by RT - PCR . caps ere treated with - ) , no injection of Co MO , Wip1 MO / or Wip1 mRNA . dose of reagent injected : Xnr1 ( 5 pg ) , Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) Wip1 ( 1 ng ) .

O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O

(A, Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps ere treated with -), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Xnr1 (5 pg), Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).


B ) Activin / Nodal - induced gene responses are enhanced further in absence of Wip1 as analyzed by RT - PCR . caps in ( B ) were treated with Activin ( 5 ng / ml ) . ( - ) , no injection of Co MO , Wip1 MO / or Wip1 mRNA . dose of reagent injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) Wip1 ( 1 ng ) .

O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O

B) Activin/Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps in (B) were treated with Activin protein (5 ng/ml). (-), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).


( C ) Knockdown of Wip1 up - regulates activity of Activin / Nodal - responsive promoter . HEK293T transfected with Co siRNA ( 50 nM ) or Wip1 siRNA ( 50 nM ) were stimulated with Activin ( 10 ng / ml ) for 10 h then subjected to luciferase assays . Data are expressed as mean ± ( n = 3 biological replicates ) . * * * P < 0 . 001 by unpaired Student ' s t - .

O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Knockdown of Wip1 up-regulates the activity of Activin/Nodal-responsive promoter. HEK293T cells transfected with Co siRNA (50 nM) or Wip1 siRNA (50 nM) were stimulated with Activin protein (10 ng/ml) for 10 h and then subjected to luciferase assays. Data are expressed as the mean ± SEM (n=3 biological replicates). ***P<0.001 by unpaired Student's t-test.


Depletion of Wip1 promotes epidermal differentiation at expense of neural fate . ( - ) , no injection of Noggin mRNA . amount of MO mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G J ; 30 ng for F I ) Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. (-), no injection of Noggin mRNA. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.


Depletion of Wip1 promotes epidermal differentiation at expense of neural fate . in ( E - J ) are shown in dorsal views with anterior to top . amount of MO mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G J ; 30 ng for F I ) Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. Embryos in (E-J) are shown in dorsal views with anterior to the top. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.


( K , Activin induction of p21 is up - regulated upon Wip1 knockdown . HEK293T were transfected with Co siRNA or Wip1 siRNA 48 h after siRNA transfection , were stimulated with Activin ( 10 ng / ml ) for 3 to 12 h as indicated t = 0 in ( K ) is 48 h after siRNA transfection . P - Smad2 serves as an indicator of active Activin / Nodal signaling . Arrows ndicate non - specific bands .

O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(K, Activin induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with Activin (10 ng/ml) for 3 to 12 h as indicated t=0 in (K) is 48 h after siRNA transfection. P-Smad2 serves as an indicator of active Activin/Nodal signaling. Arrows ndicate non-specific bands.


L ) TGF - β induction of p21 is up - regulated upon Wip1 knockdown . HEK293T were transfected with Co siRNA or Wip1 siRNA 48 h after siRNA transfection , were stimulated with increasing of TGF - β1 ( 10 , 20 ng / ml ) for 3 h .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

L) TGF-β induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with increasing concentration of TGF-β1 (10, 20 ng/ml) for 3 h.


( A , Effects of gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T were transfected or not with wt Wip1 , Wip1 ( D277A ) , as indicated subjected to CCK - 8 assays before ( 0 h ) after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - . n . s . , not significant .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with wt Wip1, Wip1(D277A), as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.


B ) Effects of gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T were transfected or not with Co siRNA or Wip1 siRNA as indicated subjected to CCK - 8 assays before ( 0 h ) after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - . n . s . , not significant .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.


Depletion of Wip1 augments TGF - β - induced migration invasion of MDA - MB231 breast cancer . MDA - MB231 transfected with Co siRNA or Wip1 siRNA were scratched with a 20 - μL pipette tip then incubated in absence or presence of TGF - β1 for indicated times ( C , D )

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20-μL pipette tip and then incubated in the absence or presence of TGF-β1 for the indicated times (C, D)


Depletion of Wip1 augments TGF - β - induced migration invasion of MDA - MB231 breast cancer . MDA - MB231 transfected with Co siRNA or Wip1 siRNA were subjected to transwell with or without TGF - β1 for 20 h ( E , F ) .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF-β1 for 20 h (E, F).


( A - D ) Knockdown of Wip1 expands expression of dorsal mesodermal markers . injected dorsally with Co MO ( 40 ng ) or Wip1 MO ( 40 ng ) were subjected to in situ hybridization at stage 10 . 25 . are shown in dorso - vegetal views with dorsal to top . Scale bar , 150 μm .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) Knockdown of Wip1 expands the expression of dorsal mesodermal markers. Embryos injected dorsally with Co MO (40 ng) or Wip1 MO (40 ng) were subjected to in situ hybridization at stage 10.25. Embryos are shown in dorso-vegetal views with dorsal to the top. Scale bar, 150 μm.


( E - I ) Morphological of Wip1 morphants . were injected radially in marginal zone with Co MO ( 80 ng ) , Wip1 MO ( 80 ng ) , wt Wip1 ( 1 ng ) Wip1 ( D277A ) mRNA as indicated cultured to tadpole stages . are shown in lateral views with anterior to left . Un Co , uninjected control . Scale bar , 1 mm . ( J ) of shown in ( E - I ) . Severe defects include microcephaly , shortened kinked axis , no eye . Mild defects indicate normal axis with malformed eyes .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-I) Morphological phenotypes of Wip1 morphants. Embryos were injected radially in the marginal zone with Co MO (80 ng), Wip1 MO (80 ng), wt Wip1 (1 ng) and Wip1(D277A) mRNA as indicated and cultured to tadpole stages. Embryos are shown in lateral views with anterior to the left. Un Co, uninjected control. Scale bar, 1 mm. (J) Quantification of the phenotypes shown in (E-I). Severe defects include microcephaly, shortened and kinked body axis, and no eye. Mild defects indicate normal body axis with malformed eyes.


( A Wip1 Smad4 form an endogenous . lysates of HEK293T , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) ( - ) , no treatment with ligand . IgG , control IgG . An arrow in ( A ) indicates non - specific bands .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) (-), no treatment with protein ligand. IgG, control IgG. An arrow in (A) indicates non-specific bands.


B ) Wip1 Smad4 form an endogenous . lysates of HEK293T , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) vice versa ( B ) . ( - ) , no treatment with ligand . IgG , control IgG .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

B) Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) and vice versa (B). (-), no treatment with protein ligand. IgG, control IgG.


( C ) Upon ligand stimulation , Wip1 forms a with Smad4 in nucleus . HeLa were treated or not with TGF - β1 ( 40 ng / ml ) for 2 h , fixed then subjected to proximity ligation assays with antibodies against Wip1 Smad4 . Blue : DAPI ; Red : PLA signal . Scale bar , 20 μm . ( D ) Quantitative of Wip1 / Smad4 interactions shown in ( C ) . Data are represented as mean ± ( n = 3 biological replicates ) . Twenty were analyzed per sample . * * * P < 0 . 001 by Student ' s t - .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Upon ligand stimulation, Wip1 forms a complex with Smad4 in the nucleus. HeLa cells were treated or not with TGF-β1 (40 ng/ml) for 2 h, fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4. Blue: DAPI; Red: PLA signal. Scale bar, 20 μm. (D) Quantitative analysis of Wip1/Smad4 interactions shown in (C). Data are represented as the mean ± SEM (n=3 biological replicates). Twenty cells were analyzed per sample. ***P<0.001 by Student's t-test.


( E ) Wip1 interferes with ability of Smad4 to bind to Smad2 . HEK293T co - transfected with indicated constructs were treated or not with TGF - β1 for 1 h then harvested . An arrow denotes Myc - tagged Wip1 .

O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O

(E) Wip1 interferes with the ability of Smad4 to bind to Smad2. HEK293T cells co-transfected with the indicated constructs were treated or not with TGF-β1 for 1 h and then harvested. An arrow denotes Myc-tagged Wip1 protein.


( F ) Wip1 has a negative effect on accumulation of Smad4 . HEK293T were transfected with Co siRNA or Wip1 siRNA , treated with TGF - β1 ( 40 ng / ml , 1 h ) 48 h after siRNA transfection subjected to / fractionation western blotting . β - tubulin lamin A / C serve as markers , respectively . C , cytoplasm ; N , nucleus . experiment was repeated two times with similar results . ( G ) of of Smad4 normalized to those of Smad4 shown in ( F ) .

O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O

(F) Wip1 has a negative effect on nuclear accumulation of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA, treated with TGF-β1 (40 ng/ml, 1 h) 48 h after siRNA transfection and subjected to nuclear/cytoplasmic fractionation and western blotting analysis. β-tubulin and lamin A/C serve as cytoplasmic and nuclear markers, respectively. C, cytoplasm; N, nucleus. The experiment was repeated two times with similar results. (G) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in (F).


( A ) Overexpression of Wip1 down - regulates Smad4 phosphorylation at Thr277 . MOCK - or Flag - Wip1 - transfected HEK293T were treated with 1 h of FGF2 ( 10 ng / ml ) , followed by addition of U0126 ( 40 μM ) harvesting for western blotting at indicated times . - FGF2 , no treatment with FGF2 .

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(A) Overexpression of Wip1 down-regulates Smad4 phosphorylation at Thr277. MOCK- or Flag-Wip1-transfected HEK293T cells were treated with 1 h pulse of FGF2 (10 ng/ml), followed by addition of U0126 (40 μM) and harvesting for western blotting at the indicated times. - FGF2, no treatment with FGF2.


( B ) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control Wip1 - depleted HEK293T , showing that knockdown of Wip1 up - regulates Smad4 phosphorylation at MAPK site . A bracket denotes phospho - Smad4 .

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(B) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1-depleted HEK293T cells, showing that knockdown of Wip1 up-regulates Smad4 phosphorylation at the MAPK site. A bracket denotes phospho-Smad4.


( C ) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase . Myc - tagged Smad4 was expressed in HEK293T , which were then treated or not with FGF2 ( 40 ng / ml , 2 h ) , Myc - Smad4 was immunoprecipitated using anti - Myc antibody subsequently incubated with or without recombinant human Wip1 ( 0 . 5 μg ) in absence or presence of Mg2 + as indicated .

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(C) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay. Myc-tagged Smad4 was expressed in HEK293T cells, which were then treated or not with FGF2 (40 ng/ml, 2 h), and the Myc-Smad4 was immunoprecipitated using anti-Myc antibody and subsequently incubated with or without recombinant human Wip1 protein (0.5 μg) in the absence or presence of Mg2+ as indicated.


( D ) FGF - induced destabilization of Smad4 is enhanced further in Wip1 - depleted cap as analyzed by western blotting . amount of reagent injected : Myc - Smad4 ( 400 pg ) , FGF8 ( 100 pg ) , Wnt8 ( 400 pg ) , Co MO ( 60 ng ) Wip1 MO ( 60 ng ) .

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(D) FGF-induced destabilization of Smad4 is enhanced further in Wip1-depleted animal cap cells as analyzed by western blotting. The amount of reagent injected: Myc-Smad4 (400 pg), FGF8 (100 pg), Wnt8 (400 pg), Co MO (60 ng) and Wip1 MO (60 ng).


( E ) Polyubiquitination of wt Smad4 is promoted in Wip1 - silenced . HEK293T were transfected with indicated combinations of Flag - ubiquitin , Co siRNA , Wip1 siRNA , Myc - Smad4 Myc - Smad4 ( T277A ) then treated with FGF2 ( 10 ng / ml , 4 h ) in presence of proteasome inhibitor MG132 , Myc - Smad4 its phosphorylation - resistant mutant were immunoprecipitated with anti - Myc antibody . Ubiquitin - conjugated Smad4 ( Smad4 - Ubn ) was detected by western blotting with anti - Flag antibody .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

(E) Polyubiquitination of wt Smad4 is promoted in Wip1-silenced cells. HEK293T cells were transfected with the indicated combinations of Flag-ubiquitin, Co siRNA, Wip1 siRNA, Myc-Smad4 and Myc-Smad4(T277A) and then treated with FGF2 (10 ng/ml, 4 h) in the presence of the proteasome inhibitor MG132, and Myc-Smad4 and its phosphorylation-resistant mutant were immunoprecipitated with anti-Myc antibody. Ubiquitin-conjugated Smad4 (Smad4-Ubn) was detected by western blotting with anti-Flag antibody.


( F ) Wip1 prolongs half - life of Smad4 . HEK293T were transfected with Co siRNA or Wip1 siRNA then treated with FGF2 along with cycloheximide ( CHX , 100 ng / ml ) for indicated times harvested for western blotting . An arrow indicates non - specific bands .

O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Wip1 prolongs the half-life of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide (CHX, 100 ng/ml) for the indicated times and harvested for western blotting. An arrow indicates non-specific bands.


( A - J ) injected dorsally with wt Wip1 ( 1 ng ) , wt Smad4 ( 1 ng ) , Smad4 ( T277A ) ( 1 ng ) Smad4 ( T277E ) ( 1 ng ) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10 . 25 . Control , control injected with LacZ mRNA only . Scale bar , 150 μm . ( K ) of results from in situ hybridization in ( A - J ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-J) Embryos injected dorsally with wt Wip1 (1 ng), wt Smad4 (1 ng), Smad4(T277A) (1 ng) and Smad4 (T277E) (1 ng) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10.25. Control, control embryo injected with LacZ mRNA only. Scale bar, 150 μm. (K) Quantification of the results from in situ hybridization analysis in (A-J).


( L ) Wip1 - mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho - mimetic acid at MAPK site . ( - ) , no co - injection of wt or mutated Smad4 .

O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

(L) Wip1-mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho-mimetic amino acid at the MAPK site. (-), no co-injection of wt or mutated Smad4.


( M ) MDA - MB231 MDA - MB468 were transfected with wt Wip1 alone or with wt Smad4 or Smad4 ( T277A ) then subjected to CCK - 8 assays to measure growth . Data are presented as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - .

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(M) MDA-MB231 and MDA-MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4(T277A) and then subjected to CCK-8 assays to measure cell growth. Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


( N ) Anchorage - independent colony - formation assays were performed in MDA - MB231 MDA - MB468 transfected with Wip1 or negative control constructs . Scale bar , 100 μm . ( O ) of assays shown in ( N ) . number of colonies larger than 50 μm in diameter was scored . Data are expressed as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - .

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(N) Anchorage-independent colony-formation assays were performed in MDA-MB231 and MDA-MB468 cells transfected with Wip1 or negative control constructs. Scale bar, 100 μm. (O) Quantification of the assays shown in (N). The number of colonies larger than 50 μm in diameter was scored. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


a - e , Scanning electron microscopy ( ) of fly eyes expressing DTS7 with or without indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . a , Normal eyes in DTS7 flies reared at 22 ° C . b , Rough eyes in DTS7 flies reared at 28 ° C . c , d , Degeneration was suppressed by expression of Drosophila HDAC6 ( dHDAC6 ; c ) or human HDAC6 ( hHDAC6 ; d ) , but not a catalytically dead mutant of hHDAC6 ( cat . mut . ; e ) .

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a-e, Scanning electron microscopy (SEM) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. a, Normal eyes in DTS7 flies reared at 22 °C. b, Rough eyes in DTS7 flies reared at 28 °C. c, d, Degeneration was suppressed by expression of Drosophila HDAC6 (dHDAC6; c) or human HDAC6 (hHDAC6; d), but not a catalytically dead mutant of hHDAC6 (cat. mut.; e).


f - j , of fly eyes expressing AR52 with or without indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . f , Normal eyes in AR52 flies reared without ( - ) DHT . g , Rough eyes in AR52 flies reared with ( + ) DHT . Degeneration was suppressed by expression of Drosophila HDAC6 ( h ) or human HDAC6 ( i ) , but not a catalytically dead mutant of human HDAC6 ( j ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

f-j, SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. f, Normal eyes in AR52 flies reared without (-) DHT. g, Rough eyes in AR52 flies reared with (+) DHT. Degeneration was suppressed by expression of Drosophila HDAC6 (h) or human HDAC6 (i), but not a catalytically dead mutant of human HDAC6 (j).


k - p , Detection of UPS reporter in imaginal eye discs from third - instar larvae by confocal microscopy . High level was found in flies expressing GFP ( k , positive control ) , but was barely detectable in control flies expressing CL1 - GFP ( l , negative control ) . CL1 - GFP accumulates in DTS7flies with - dependent proteasome impairment ( compare m to n ) in AR52flies with ligand - dependent degeneration ( compare o to p ) . retinal of 200 to > 1 , 000 flies of each genotype were examined . Quantitative analyses of eye proteasome impairment are presented in Supplementary Figs S2 S3 , respectively . ( DHT , dihydrotestosterone . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

k-p, Detection of UPS reporter in imaginal eye discs from third-instar larvae by confocal microscopy. High level fluorescence was found in flies expressing GFP (k, positive control), but fluorescence was barely detectable in control flies expressing CL1-GFP (l, negative control). CL1-GFP accumulates in DTS7flies with temperature-dependent proteasome impairment (compare m to n) and in AR52flies with ligand-dependent degeneration (compare o to p). The retinal phenotypes of 200 to >1,000 flies of each genotype were examined. Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3, respectively. (DHT, dihydrotestosterone.)


a - e , Representative examples of autophagic vacuoles detected by in retinal sections used to generate quantitative data shown in ( f ) . a , An autophagosome ( red arrow ) containing contents in a photoreceptor neuron from an AR52fly reared on DHT . b , Higher magnification of autophagosome in a . c , Multiple autophagolysosomes ( red arrows ) containing dense , amorphous material from an AR52fly reared on DHT . d , A juxtanuclear multilamellar ( red arrow ) from a DTS7fly reared at 28 ° C . e , A multivesicular ( red arrow ) from a DTS7fly reared at 28 ° C . f , A significant increase in frequency of neurons with autophagic figures in DTS7 flies reared at 28 ° C compared to those reared at 22 ° C , in AR52 flies reared on DHT compared to those reared off DHT . Data show mean ± s . d . , n = 59 - 82 neurons in 5 sections per condition . No accumulation of autophagic figures was found in AR12 flies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O

a-e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f). a, An autophagosome (red arrow) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT. b, Higher magnification of the autophagosome in a. c, Multiple autophagolysosomes (red arrows) containing dense, amorphous material from an AR52fly reared on DHT. d, A juxtanuclear multilamellar body (red arrow) from a DTS7fly reared at 28 °C. e, A multivesicular body (red arrow) from a DTS7fly reared at 28 °C. f, A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 °C compared to those reared at 22 °C, and in AR52 flies reared on DHT compared to those reared off DHT. Data show mean ± s.d., n = 59-82 neurons in 5 sections per condition. No accumulation of autophagic figures was found in AR12 flies.


g - l , of fly eyes expressing indicated transgenes . RNAi knockdown ( KD ) of atg6 atg12 enhances degeneration in DTS7 flies reared at 28 ° C ( compare h , i to g ) AR52 flies reared on DHT ( compare k , l to j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye are presented in Supplementary Fig . S2 . ( N , nucleus ; Rh , rhabdomere . )

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

g-l, SEM images of fly eyes expressing the indicated transgenes. RNAi knockdown (KD) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 °C (compare h, i to g) and AR52 flies reared on DHT (compare k, l to j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2. (N, nucleus; Rh, rhabdomere.)


a , b , d , Western blots from flies expressing indicated transgenes . a , Steady state of AR52 are reduced in flies overexpressing Drosophila HDAC6 , but are elevated in flies in which atg6 or atg12 has been knocked down . b , Western blots showing temporal profile of AR52 monomer high molecular aggregate after a brief of expression . AR52 became detectable by 2 . 5 h after treatment with RU486 , reached a peak at 10 h , then slowly decayed . c , A logarithmic plot of AR52 / actin ratios was used to determine of best fit by regression ( y = Ae - Kx ) . R2 = 0 . 9117 ( AR52 - DHT ) , R2 = 0 . 7808 ( AR52 + DHT ) , R2 = 0 . 9719 ( HDAC6 + ( AR52 - DHT ) ) , R2 = 0 . 9644 ( HDAC6 + ( AR52 + DHT ) ) . Half - life was determined by slope of best fit with equation t1 / 2 = 0 . 693 / K . Half - life of AR52 in vivo was reduced ∼ 2 - fold in flies co - expressing Drosophila HDAC6 did not differ significantly in presence ( broken lines ) or absence ( solid lines ) of DHT . Plots of mean AR52 / actin ratios are shown in Supplementary Fig . S12 . d , Flies co - expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b , but AR decayed at an accelerated rate . Exogenous Drosophila HDAC6 was detected by immunoblot against V5 epitope .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O

a, b, d, Western blots from flies expressing the indicated transgenes. a, Steady state levels of AR52 protein are reduced in flies overexpressing Drosophila HDAC6, but are elevated in flies in which atg6 or atg12 has been knocked down. b, Western blots showing the temporal profile of AR52 protein monomer and high molecular weight aggregate levels after a brief pulse of expression. AR52 protein became detectable by 2.5 h after treatment with RU486, reached a peak at 10 h, and then slowly decayed. c, A logarithmic plot of AR52/actin ratios was used to determine the line of best fit by regression analysis (y = Ae-Kx). R2 = 0.9117 (AR52 - DHT), R2 = 0.7808 (AR52 + DHT), R2 = 0.9719 (HDAC6 + (AR52 - DHT)), R2 = 0.9644 (HDAC6 + (AR52 + DHT)). Half-life was determined by the slope of the best fit line with the equation t1/2 = 0.693/K. Half-life of AR52 in vivo was reduced ∼2-fold in flies co-expressing Drosophila HDAC6 and did not differ significantly in the presence (broken lines) or absence (solid lines) of DHT. Plots of the mean AR52/actin ratios are shown in Supplementary Fig. S12. d, Flies co-expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b, but AR protein decayed at an accelerated rate. Exogenous Drosophila HDAC6 was detected by immunoblot against the V5 epitope.


a - l , of fly eyes expressing indicated transgenes . rough eye caused by proteasome mutation ( a ) or by expression of polyQ - expanded AR ( b ) were both suppressed by rearing flies on TOR inhibitor rapamycin ( c , d ) . e , f , Rapamycin failed to suppress degeneration in an autophagy - deficient background created by knockdown of atg12 , confirming that rescue by rapamycin is autophagy - dependent . g , h , Rapamycin also failed to suppress degeneration when HDAC6 were knocked down , demonstrating that suppression via TOR pathway is HDAC6 - dependent . k , j , HDAC6 failed to suppress degeneration in an autophagy - deficient background , confirming that rescue by HDAC6 is dependent on autophagy ( compare k , l to i , j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye are presented in Supplementary Fig . S2 .

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a-l, SEM images of fly eyes expressing the indicated transgenes. The rough eye phenotypes caused by proteasome mutation (a) or by expression of polyQ-expanded AR (b) were both suppressed by rearing flies on the TOR inhibitor rapamycin (c, d). e, f, Rapamycin failed to suppress degeneration in an autophagy-deficient background created by knockdown of atg12, confirming that rescue by rapamycin is autophagy-dependent. g, h, Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down, demonstrating that suppression via the TOR pathway is HDAC6-dependent. k, j, HDAC6 failed to suppress degeneration in an autophagy-deficient background, confirming that rescue by HDAC6 is dependent on autophagy (compare k, l to i, j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2.


A - L HeLa stably expressing FLAG - tagged UNC13D ( F - UNC13D ) ( A B ) , UNC13D stable knock - down ( KD ) ( C , E , G , I K ) or knock out ( KO ) ( D , F , H , J L ) HeLa , their control were infected with VACV ( A , C D ) , HSV ( E F ) , or SeV ( K L ) , or they were transfected with plasmid ( pDNA ) ( 1 μg / mL ) ( B , G - J ) for indicated time , before immunoblotting with anti - Flag , - UNC13D , - p - TBK1 , - p - IRF3 , - STING , - p - P65 , - p - IκB - α , - actin or - tubulin antibody .

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A - L HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (A and B), UNC13D stable knock-down (KD) (C, E, G, I and K) or knock out (KO) ( D, F, H, J and L) HeLa cells, and their control cells were infected with VACV (A, C and D), HSV (E and F), or SeV (K and L), or they were transfected with plasmid DNA (pDNA) (1 μg/mL) (B, G-J) for the indicated time, before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -p-P65, -p-IκB-α, -actin or -tubulin antibody.


UNC13D stable knock - down ( KD ) HeLa their control were transfected with plasmid ( 1 μg / mL ) for indicated time . UNC13D , IFN - β IL6 mRNA were measured by qPCR ( M - O ) .

B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

UNC13D stable knock-down (KD) HeLa cells and their control cells were transfected with plasmid DNA (1 μg/mL) for the indicated time. UNC13D, IFN-β and IL6 mRNA levels were measured by qPCR (M-O).


UNC13D stable knock - down ( KD ) HeLa their control were transfected with plasmid ( 1 μg / mL ) infected with SeV ( Q ) for indicated time . secretion of IFN - β was measured by bioassay ( P Q ) .

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UNC13D stable knock-down (KD) HeLa cells and their control cells were transfected with plasmid DNA (1 μg/mL) infected with SeV (Q) for the indicated time. The secretion of IFN-β was measured by bioassay (P and Q).


R , S UNC13D knock - out ( KO ) HeLa their control were infected with GFP - HSV for 18 h or 24 h . GFP expression was visualized ( left ) green was determined by ImageJ .

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R, S UNC13D knock-out (KO) HeLa cells and their control cells were infected with GFP-HSV for 18 h or 24 h. GFP expression was visualized (left) and the green fluorescence intensity was determined by ImageJ.


A HEK293T were transfected with indicated plasmids ( 5 μg each ) for 24 h . Whole lysates ( WCL ) were examined , lysates were subjected to immunoprecipitation ( IP ) with anti - HA antibodies , followed by immunoblotting ( IB ) with anti - HA - Flag antibody .

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A HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (IB) with anti-HA and -Flag antibody.


B - E HeLa stably expressing FLAG - tagged UNC13D ( F - UNC13D ) ( B ) , UNC13D stable knock - down ( KD ) ( D ) or knock - out ( KO ) ( C E ) HeLa , their control were stimulated with cGAMP ( 100 nM ) for 30 min ( B C ) or for indicated time ( D E ) before immunoblotting with anti - Flag , - UNC13D , - p - TBK1 , - p - IRF3 , - STING , - GAPDH , - actin or - tubulin antibody .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O

B - E HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (B), UNC13D stable knock-down (KD) (D) or knock-out (KO) (C and E) HeLa cells, and their control cells were stimulated with cGAMP (100 nM) for 30 min (B and C) or for the indicated time (D and E) before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -GAPDH, -actin or -tubulin antibody.


F - H HEK293T were transfected with indicated plasmids ( 5 μg each ) for 24 h ( F G ) . UNC13D knock - out ( KO ) control HeLa were stimulated with cGAMP ( 100 nM ) for 30 min ( H ) . Whole lysates ( WCL ) were examined , lysates were immunoprecipitated ( IP ) with anti - HA antibodies ( F G ) , IgG anti - STING antibodies ( H ) , followed by immunoblotting ( IB ) with anti - HA , - Flag antibody or indicated antibodies .

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F- H HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h (F and G). UNC13D knock-out (KO) and control HeLa cells were stimulated with cGAMP (100 nM) for 30 min (H). Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies (F and G), IgG and anti-STING antibodies (H), followed by immunoblotting (IB) with anti-HA, -Flag antibody or the indicated antibodies.