EMBO/soda-nel huggingface dataset:
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 (2019)
Figure 1

Figure 1. RNF8 is a ubiquitinated substrate of p97 under physiological and genotoxic conditions

Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA-mediated p97 depletion under physiological conditions and after IR (10 Gy).

Graph represents the quantifications of (A) (***P<0.001; unpaired t-test, n=3, mean +SEM).

Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10µM for 6h) under physiological conditions and after IR (10 Gy).

Graph represents the quantifications of (C) (*P<0.05, ****P<0.0001; unpaired t-test, n=3, mean +SEM).

Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline-induced mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy).

Graph represents the quantifications of (E) (**P<0.01, ****P<0.0001; unpaired t-test, n=4, mean +SEM).

Model representing the processing of ubiquitinated substrate by p97 ATPase activity. Inactivation of p97 ATPase activity leads to the accumulation of ubiquitinated substrate.

Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA-mediated p97 depletion.

Graph represents the quantifications of (H) (***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM) and Western blot for efficacy of siRNA depletion of p97 (right).

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing K48-linked hyper-ubiquitination of RNF8 after siRNA-mediated p97 depletion.

Western blot analysis of Strep-p97 Co-IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR (10 Gy).

Graph represents the quantifications of (K) (*P<0.05; unpaired t-test, n=2, mean +SEM).
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 (2019)
Figure 2

Figure 2. Homeostasis of RNF8 is controlled by auto-ubiquitination and the ubiquitin proteasome system

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing the ubiquitination pattern of RNF8-WT and RNF8-RING* variant, under siRNA-mediated luciferase (siLuc) or p97-depleted conditions (sip97).

Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10µM).

Graph represents the quantifications of (B) (ns; not significant, P>0.05, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing hyper-ubiquitination of RNF8 after proteasome inhibition (MG132, 10µM for 6h).

Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag-RNF8-WT and Flag-RNF8-RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8.

Graph represents the quantifications of (E) (ns P>0.05, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 (2019)
Figure 3

Figure 3. Ataxin-3 is a nuclear p97 associated DUB that interacts with RNF8

Schematic representation of a SILAC-based mass-spectrometry approach (upper graph). The p97 interactome showing ATX3 as the only DUB in nucleus with increased interaction with p97 after IR (10 Gy; lower graph).

Schematic representation of ATX3 domain structure. C14; cysteine catalytic centre at amino-acid 14, UBS1 or 2; ubiquitin binding sites 1 or 2, UIM; ubiquitin interacting motif, VBM; p97 binding motif, poly Q; polyglutamine region.

Western blot analysis of Strep-p97 Co-IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR (10 Gy).

Western blot analysis of Flag-ATX3 Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and PSα6 under physiological conditions and after IR (10 Gy).

Western blot analysis of Flag-ATX3 WT or Flag-ATX3 VBM* Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and p97 core co-factors Npl4-Ufd1. (* represents unspecific bands).

Western blot analysis of Strep-p97EQ Co-IP in HEK293 cells, in presence or absence (CRISPR knockout; ∆ATX3) of ATX3, showing increased interaction of RNF8 with p97 in absence of ATX3, under physiological conditions and after IR (10 Gy).

Graph represents quantifications of (F) (*P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).

Model showing the interaction of p97 and ATX3 with ubiquitinated RNF8.
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 (2019)
Figure 4

Figure 4. ATX3 deubiquitinates RNF8

Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ∆ATX3 cell extract. Arrow represents the main RNF8 band and asterisks represents unspecific bands.

Graphs represent the quantifications of (A). RNF8 level at starting point (0h) was shown without equalisation (left). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate (right). (**P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).

Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ATX3 knockdown HeLa cells. The degradation was completely blocked after simultaneous inhibition of proteasome (MG132, 10µM).

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing RNF8 hyper-ubiquitination in siRNA-mediated p97 or ATX3-depleted conditions.

Western blot analysis of Flag-RNF8 denaturing-IP in HeLa cells showing RNF8 hyper-ubiquitination in ∆ATX3 condition. RNF8 hyper-ubiquitination was suppressed by reintroduction of GFP-ATX3-WT but not with GFP-ATX3-C14A or GFP-ATX3-UIM*.
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 (2019)
Figure 5

Figure 5. ATX3 protects RNF8 from premature degradation

Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm+nucleoplasm) of HeLa cells, expressing DOX-inducible GFP-ATX3-C14A variant as compared to GFP-ATX3-WT. Endogenous ATX3 was depleted with siRNA targeting 3'UTR region of ATXN3.

Graph represents the quantifications of (A). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (*P<0.05, **P<0.01; two-way ANOVA, n=2, mean +SEM).

Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm + nucleoplasm) of HeLa cells, expressing DOX-inducible ATX3-UIM* variant as compared to ATX3-WT. Endogenous ATX3 was depleted with siRNA ATX3_3'UTR.

Graph represents the quantifications of (C). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (ns P>0.05, *P<0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).

Western blot analysis showing endogenous RNF8 protein level in soluble fraction (Cytosol and Nucleosol) of HeLa cells under indicated conditions. RNF8 level was significantly reduced after siRNA mediated ATX3 depletion and then was significantly rescued by DOX-inducible expression of GFP-ATX3-WT but not with GFP-ATX3-VBM.

Graphs represent the quantifications of (E). (ns P>0.05, * P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).

Representative IF images showing γH2AX-positive micronuclei (white arrows) under physiological conditions with indicated siRNA mediated depletion. (Scale bar represents 10 µm)

Graph represents quantification of (G), showing percentage of cells with γH2AX-positive micronuclei in U2OS cells. (* P<0.05, **P<0.01, ***P<0.001; unpaired t-test, n=3, mean +SEM, at least 200 randomly selected nuclei were counted per condition per experiment).
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 (2019)
Figure 6

Figure 6. The p97-ATX3 complex suppresses hyper-accumulation of RNF8 and K63-Ub at sites of DNA damage

Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA-depleted conditions, before and after IR (4 Gy). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).

Colony forming assay (CFA) showing the IR sensitivity of HeLa cells under siRNA-mediated p97 or ATX3-depleted conditions. (n=3, mean ±SD).

CFA showing the IR sensitivity of HeLa-WT cells compared to HeLa∆ATX3 cells. (n=3, mean ±SD).

CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-C14A or ATX3-UIM* variant. (ns P>0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).

CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-VBM variant. (ns P>0.05, *P<0.05, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).

Representative IF micrographs in U2OS cells showing IRIF formation of GFP-RNF8-WT, 53BP1 and γH2AX (first panel) and GFP-RNF8-∆RING, 53BP1 and γH2AX (second panel) under indicated siRNA-depleted or p97-inhibited (CB5083) conditions. (Scale bar; 10µm).

Quantification of (F) for GFP-RNF8-WT. Graph represents the percentage of cells with increased GFP-RNF8-WT foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).

Quantification of (F) for GFP-RNF8-∆RING. Graph represents the percentage of cells with increased GFP-RNF8-∆RING foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (ns P>0.05, *P<0.01, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).

Representative IF images and quantification showing endogenous K63-Ub and γ-H2AX at UV-A micro-laser-induced DNA damage tracks in U2OS cells under indicated siRNA-depleted conditions. (Scale bar; 10µm).

Quantification of (I). Graph represents the average intensity of the K63-Ub (ns P>0.05, **P<0.01, ***P<0.001; unpaired t-test, n=2, mean +SEM, more than 50 nuclei were analysed).
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 (2019)
Figure 7

Figure 7. The p97-ATX3 complex fine-tunes histone H1 ubiquitination after DSBs

Representative IF images showing the average intensity of endogenous RNF168 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).

Graph represents the quantification of (A). (***P<0.001; unpaired t-test, n=3, mean +SEM, more than 50 nuclei were analysed).

Representative IF images showing the average intensity of endogenous 53BP1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).

Graph represents the quantification of (C). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 80 nuclei per condition and experiment).

Western blot analysis of Flag-histone H1 denaturing-IP, showing ubiquitination pattern of histone H1 after 30 min of IR (10 Gy) treatment under indicated siRNA-depleted conditions.

Representative IF images showing the average intensity of endogenous BRCA1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).

Graph represents the quantification of (F). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 100 nuclei per condition and experiment).
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 (2019)
Figure 8

Figure 8. The p97-ATX3 complex promotes accurate execution of NHEJ and DSB repair

GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (*P<0.05, **P<0.01, ***P<0.001; one-way ANOVA, n=3, mean +SEM).

GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (ns P>0.05, **P<0.01; one-way ANOVA, n=3, mean +SEM).

CFA showing the IR sensitivity of 53BP1-proficient and deficient MCF7 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (ns P>0.05; two-way ANOVA, n=2, mean ±SEM).

CFA showing the IR sensitivity of BRCA2-proficient and deficient DLD1 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (**P<0.01, ***P<0.001; two-way ANOVA, n=2, mean ±SEM).

Representative IF-images showing Rad51 foci in S-phase (Edu positive) U2OS nucleus after 5h of IR (2Gy) exposure. (Scale bar; 10µm).

Graph represents the quantification of (E) showing average number of Rad51 foci per S-phase (Edu positive) nucleus. (ns P>0.05, ****P<0.0001; unpaired t-test, n=2, mean +SEM, at least 50 nuclei per condition and experiment).

CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after co-depletion (siRNA) of RNF8. (ns P>0.05, *P<0.05, ****P<0.0001; two-way ANOVA, n=3, mean ±SEM).

Model representing p97-ATX3 complex maintaining RNF8 homeostasis under soluble environment and promoting RNF8 chromatin extraction at sites of DSBs to promote NHEJ.

Expanded View Figure Legends
WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation (2017)
Figure 1

Figure 1. Dendritic actin networks can grow faster than individual filaments under the same biochemical conditions. (A) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin-actin in solution. (B) Time-lapse TIRF microscopy of single filament growth from Alexa488 phalloidin-stabilized seeds (green) in the presence of 5μM profilin-actin. (C) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF- and lipid-coated microspheres with profilin-actin, CP and Arp2/3 in solution. (D) Widefield epifluorescence images of indicated network components of bead-attached dendritic networks after kinetic arrest at indicated times. Biochemical conditions are matching (B) with the exception of the additional presence of 200nM CP and 50nM Arp2/3 (E) Scheme of dendritic network growth from square WAVE1-micropatterns on a 2-dimensional coverslip with profilin-actin, CP and Arp2/3 in solution. (F) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth. Conditions are as in (D) (G) Average length of individual filaments (red, N=15), bead- (green, N=28-48 per time point) or NPF micropattern-assembled (blue, N=20) dendritic networks as a function of time. (H) Distributions of growth velocities for individual filaments (red, N=125), bead- (green, N=191) or NPF micropattern- (blue, N=65) assembled dendritic networks. All error indicators are SD.
WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation (2017)
Figure 2

Figure 2. NPFs promote filament elongation locally. (A) Scheme of actin filaments growing on functionalized glass surfaces containing WAVE1 micropatterns in the presence of profilin-actin in solution. (B) TIRF microscopy of actin monomers (blue, 1uM total, 30% Cy5 labeled) in the presence of equimolar profilin recruited to a WAVE1 pattern (magenta). (C) TIRF microscopy of actin polymerization in- (blue arrows) and outside (red arrows) of WAVE1-micropatterns (magenta) in the presence of 1uM profilin-actin visualized by a filament-binding probe (5nM Alexa488-UTRNN). (D) Kymograph of a growing filament barbed end entering a WAVE1 micropattern. Conditions as in (C) (E) Distributions of barbed end growth velocities for filaments growing outside (green, N=115) or inside (magenta, N=102) of WAVE1-micropatterns compared to a negative control (non-NPF treated PEG surface, black, N=61) (F) Left: Barbed end growth velocities (dots) in 1uM profilin-actin as a function of measured mCherry-WAVE1 surface density. NPF surface density was varied using varying amounts of dark mCherry competitor (see Methods). The red line is a linear fit to the data. Error bars are SD. Right: Surface mCherry-WAVE1 fluorescence and corresponding kymographs of barbed end growth for indicated examples. (G) Surface mCherry-NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1, N-WASP or dark mCherry control constructs in 1uM profilin-actin. (H) Barbed end growth velocities for indicated WAVE1, N-WASP or dark mCherry control constructs in 1uM profilin-actin.
WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation (2017)
Figure 3

Figure 3. NPFs can stimulate filament growth by recruiting monomers to their WH2 domain. (A) Domain architecture and boundaries for WAVE1 fragments used in this study. (B) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of actin polymerization on WAVE1-coated coverslips. Conditions are 1uM actin, 5nM Alexa488-UTRNN. (C) Average barbed end growth velocities of actin polymerization on WAVE1-immobilized surfaces. At least 40 filaments were analyzed per condition. (D) Scheme of actin polymerization from NPF-actin complexes in solution. (E) TIRF microscopy of actin polymerization from 2uM actin in the absence (upper panel) or presence of near-saturating amounts of WAVE1 WCA. (F) Barbed end polymerization rates as a function of actin and WAVE1-actin ratios. The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on-rates as indicated (see Methods).
WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation (2017)
Figure 4

Figure 4. Profilin shunts NPF WH2 domains to barbed ends where they inhibit filament growth. (A) Scheme of the domain architecture of WAVE1 variants immobilized to lipid-coated beads. Nucleation is always driven from wild-type WAVE PWCA (60% total), while other variants (40% total) do not activate the Arp2/3 complex (Figure EV 4A,B) but can affect filament elongation (B) Widefield epifluorescence images (Alexa488-actin) of bead-attached dendritic networks following kinetic arrest after 10 min of network growth for spike-in experiments (i.e. 60% his10-darkCherry-PWCA, 40% his10-Cherry-W; molar percentage) using indicated WAVE1 variants. Conditions are 5μM profilin-actin, 200nM CP and 50nM Arp2/3. For all conditions, actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction. (C) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control (N= 57-73 beads per condition). Errors bars represent SD. (D) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of polymerization from profilin-actin on WAVE1-coated coverslips. Conditions are 2µM actin, 3µM profilin, 5nM Alexa488-UTRNN. (E) Average barbed end polymerization rates for indicated WAVE1 variants. Errors bars represent SD.
WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation (2017)
Figure 5

Figure 5. Monomer shuttling from the PRD regulates the monomer occupancy of the WH2 domain. (A) The weighted mass average of WAVE1 PRD (15 μM) determined by sedimentation equilibrium ultracentrifugation with LatB-stabilized monomers (15 μM) and various profilin concentrations as indicated. The lower dashed line indicates the expected mass of WAVE1 PRD; the 5-7 kDa upshift is due to non-specific actin binding (WAVE1 PRD[null], black symbols). The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD[B] in the presence of 90 μM profilin alone. (B) Experimental scheme of detecting WH2 occupancy by FRET. (C) FRET efficiencies for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P_CA=grey) as a function of LatB-stabilized, quencher- (Atto540Q-) labeled actin monomers concentration. Lines are fits to a single site binding curve. (D) WH2 occupancies (left axis, Methods) and FRET efficiencies (right axis) for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P*WCA(null)=cyan, P_CA=grey) as a function of profilin concentration. Concentration of LatB-stabilized, Atto540Q-actin monomers was constant (175nM) at all conditions. Lines are fits to a competitive inhibition model (see Methods).
SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes (2016)
Figure 3

Figure 3. Quantitative analysis of TNF-RSC-associated proteins identifies SPATA2 as a novel component of the TNF-RSC.

A. Strategy for quantification of TNF-RSC-associated proteins. Cells were treated with FLAG-TNF-α for 5 and 15 minutes, and in control FLAG-TNF-α was added directly in the lysates. The TNF-RSCs were affinity-purified using anti-FLAG antibody, proteins were mixed, proteolysed and the relative abundance of proteins quantified with LC-MS/MS.

B. Validation of the TNF-RSC interacting proteins. A549 cells were stimulated with FLAG-TNF-α for the indicated time and the TNF-RSCs were isolated as described in Figure 3A. TNF-α-stimulation-dependent interaction of the indicated proteins was confirmed by isolating the TNF-RSC and blotting with the indicated antibodies. The amount of the analyzed proteins in the input material used for the TNF-RSC isolation is also shown.

C. Quantification of ubiquitin linkage peptides in the TNF-RSC. The box plot shows the relative abundance of K48, K63 and linear ubiquitin linkage-specific peptides in the TNF-RSCs isolated from TNF-α-stimulated (15 min) and control cells quantified by SILAC-based MS.

D. Strategy for identification of SPATA2 interacting proteins. SILAC-labeled A549 cells were transfected with the GFP-SPATA2 or GFP alone, and one of GFP-SPATA2 transfected cell population was left unstimulated and the other one was stimulated with TNF-α for 15 minutes. Proteins were affinity-purified using GFP-Trap agarose and analyzed by LC-MS/MS.

E. The list of proteins enriched in SPATA2 pull-down. The table shows proteins that were enriched in GFP-SPATA2 pull-down. The columns under “Unstimulated” shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from unstimulated cells, whereas the columns under “Stimulated (TNF-α 15 min)” shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from cells stimulated with TNF-α for 15 minutes.

F. Validation of TNF-α-stimulation-independent association of SPATA2 with CYLD. GFP-SPATA2 was immunoprecipitated from unstimulated and TNF-α-stimulated cells, and probed with CYLD antibody.

G. HOIP interacts with SPATA2. FLAG-HOIP expressing A20.2J cells were SILAC labeled and FLAG-HOIP was immunoprecipitated from unstimulated cells and associated proteins were quantified using LC-MS/MS. The table contains proteins that were enriched in HOIP pull-down compared to control pull-down.

H. Validation of SPATA2 interaction with HOIP. GFP-tagged RNF31 was purified from unstimulated or TNF-α-stimulated (15 min) HCT116 cells and the immunoprecipitates were blotted with SPATA2, CYLD and GFP (HOIP) antibodies. The lower panels show expression of the indicated proteins in the input. The asterisk indicates unspecific band.