EMBO/soda-nel huggingface dataset:
Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB (2022)
Figure 5

Fig. 5 ARL11 requires PLD1 to promote phagocytosis

(A) ARL11 expression is enriched in macrophages. Analysis of single-cell RNA sequencing dataset shows that ARL11 expression is highest in macrophages (https://www.proteinatlas.org/ENSG00000152213-ARL11/tissue+cell+type).

(B) ARL11 Knockout of THP-1 stable cell pools. ARL11 knockout THP-1 cell pools were generated by the CRISPR/Cas9 technology with three different guide RNAs. Cell lysates were immunoblotted with the indicated antibodies.

(C) ARL11 knockout inhibits phagocytosis. Macrophages were differentiated from THP1 cell pools, followed by incubation with green zymosan particles for 1.5 hours. Free green zymosan particles were washed away before photography. Details were described in the methods. Typically, all cells and cellular particles in randomly selected fields were counted. Data are presented as mean± SD, n = 5 technically independent samples. One way ANOVA; *P < 0.05.

(D) THP-1 cell pool stably expressing HA-tagged ARL11Q67L or ARL11T26N. Lysates were immunoblotted with indicated antibodies.

(E) ARL11Q67L, but not ARL11T26N, promotes phagocytosis of macrophages. Macrophages were differentiated from indicated THP1 cell pool, followed by incubated with green zymosan particles for 1 hour. Scale bars: 50 μm. The result is a representative of two biologically independent experiments.

(F) Quantification result of (E). Data are presented as mean± SD, n = 8 technically independent samples. One way ANOVA; *P < 0.05.

(G) PLD1 knockout efficiency and HA-tagged ARL11Q67L expression of THP-1 stable cell pools. Lysates were immunoblotted with indicated antibodies.

(H) PLD1 knockout blocks the ARL11Q67L-stimulated phagocytosis. Macrophages were differentiated from indicated THP1 cell groups and then incubated with green zymosan particles for 1.5 hours. Scale bars: 50 μm. The result is a representative of two biologically independent experiments.

(I) PLD1 is required for ARL11 to stimulate phagocytosis. Data are quantification of panel (H). Data are presented as mean± SD, n = 5 technically independent samples. Two-way ANOVA; *P < 0.05.
An exon junction complex-independent function of Barentsz in neuromuscular synapse growth (2021)
Figure 1

Figure 1: Interaction with the EJC is required for Btz function in the ovary. (A) diagram of the genomic rescue constructs in which wild type Btz (Btz-WT) or Btz with the H215A and D216A mutations (Btz-HD) is C-terminally tagged with GFP and driven by its endogenous regulatory sequences. (B) Co-immunoprecipitation of HA-tagged Btz-WT or Btz-HD with Myc-tagged GFP or eIF4AIII from transfected S2R+ cells using Myc-Trap Magnetic Agarose beads. The upper Western blot is blotted with anti-Myc and the lower with anti-HA. Input samples are shown on the left and immunoprecipitates on the right. Btz-WT co-immunoprecipitates with eIF4AIII, but Btz-HD does not, although the protein is equally stable. The asterisk represents a likely cleavage product of Btz. (C-F) show stage 10 egg chambers stained with anti-Vasa (C-F) or anti-GFP (E', F'). (C) wild type; (D) germline clones homozygous for the null allele btz2; (E) btz2 germline clones rescued with Btz-WT; (F) btz2 germline clones rescued with Btz-HD. Posterior is to the right. Scale bars, 80 μm. Vasa localization to the posterior pole of the oocyte is lost in btz mutants and is rescued by the wild type but not the EJC interaction-defective transgene. Wild-type Btz-GFP, but not the mutant form, is also slightly enriched at the posterior pole.
An exon junction complex-independent function of Barentsz in neuromuscular synapse growth (2021)
Figure 2

Figure 2: Btz acts as an EJC component to control the distribution of mitochondria in muscle. (A-K) Confocal xz-sections of larval muscle 6 in segment A3 stained with anti-ATP5A to label mitochondria. The dorsal surface (D) is to the right and ventral (V) to the left. (A) white (w)1118 control; (B) btz2/+ heterozygote; (C) btz2/ Df(3R)BSC497; (D) G14-GAL4/UAS-btz; btz2/ Df(3R)BSC497; (E) Btz-WT; btz2/ Df(3R)BSC497; (F) Btz-HD; btz2/ Df(3R)BSC497; (G) C57-GAL4/+ control; (H) C57-GAL4>UAS-btz RNAi; (I) C57-GAL4>UAS-mago RNAi; (J) C57-GAL4>UAS-eIF4AIII RNAi; (K) C57-GAL4>UAS-blw RNAi. In wild type muscles, mitochondria are present at the dorsal surface (red arrow in A) and also distributed between the fibers (yellow arrow in A), while in btz mutant muscles and in muscles expressing btz, mago or eIF4AIII RNAi they are concentrated at the dorsal surface. Expression of UAS-btz in muscle or the presence of the wild type, but not the HD mutant, Btz transgene restores the wild type distribution to btz mutants. The loss of ATP5A staining in muscles in which blw is knocked down confirms that the signal is specific. (L-N) electron micrographs showing cross-sections of larval muscle 6 in segment A3, with the dorsal surface to the right. Scale bars, 10 μm. (L) btz2/+; (M) Df(3R)BSC497/+; (N) btz2/ Df(3R)BSC497. Yellow arrows indicate examples of mitochondria interspersed between muscle fibers, and red arrows indicate mitochondria at the dorsal surface. Scale bars, 2 μm. (O) quantification of the ratio of the dorsal surface peak of ATP5 intensity to the second highest peak for the genotypes indicated. ***, p<0.001; **p<0.01; *p<0.05; ns, not significant by Mann-Whitney test. n=9 (btz/+), n=11 (Df/+, btz/Df), n=6 (btz/Df; G14>btz, C57-GAL4/+), n=4 (btz/Df; Btz-WT, C57>eIF4AIII RNAi), n=5 (btz/Df; Btz-HD), n=10 (C57>btz RNAi), or n=3 (C57>mago RNAi). Error bars show mean ± SEM. (P) Western blot of wild type (Canton S) and btz2/Df(3R)BSC497 larval carcasses with anti-ATP5A and anti-tubulin. Duplicate samples of 15 ng total protein are shown for each genotype. The overall level of ATP5A is not altered in btz mutant muscles.
An exon junction complex-independent function of Barentsz in neuromuscular synapse growth (2021)
Figure 3

Figure 3: Btz acts in muscles, motor neurons and glia to control NMJ size. (A-J) confocal images of the NMJ on larval muscles 6 and 7 in segment A3, stained with anti-HRP (magenta) to label the nerve and anti-Dlg (green) as a postsynaptic marker of synaptic boutons. (A) w1118 control; (B) btz2/+; (C) btz2/Df(3R)BSC497; (D) UAS-btz/+; da-GAL4, btz2/Df(3R)BSC497; (E) BG380-GAL4/+; UAS-btz/+; btz2/Df(3R)BSC497; (F) 24B-GAL4/UAS-btz; btz2/Df(3R)BSC497; (G) BG380-GAL4/+; 24B-GAL4/UAS-btz; btz2/Df(3R)BSC497; (H) BG380-GAL4/+; UAS-btz RNAi/+; (I) mhc-GAL4/UAS-btz RNAi; (J) repo-GAL4/UAS-btz RNAi. Scale bar, 30 μm. Insets in (A, C) show enlargements of the boxed regions, illustrating the closer spacing of boutons in btz mutants. (K, L) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001; **, p<0.01; ns, not significant by unpaired t-test with Welch's correction. n=23 (w; da-GAL4, btz/Df), n=6 (btz/+), n=10 (Df/+, UAS-BtzGFP/+; btz/Df), n=8 (btz/Df), n=22 (da>btz-GFP; btz/Df), n=13 (BG380-GAL4; btz/Df; BG380+24B>Btz-GFP; btz/Df; da-GAL4/+), n=5 (BG380>Btz-GFP; btz/Df), n=11 (24B-GAL4; btz/Df; da>btz RNAi), n=12 (24B>Btz-GFP; btz/Df; btz RNAi/+), n=9 (BG380+24B-GAL4; btz/Df), n=15 (BG380-GAL4/+), n=14 (BG380>btz RNAi; repo>btz RNAi), n=16 (mhc-GAL4/+), n=28 (mhc>btz RNAi), or n=21 (repo-GAL4/+). Error bars show mean ± SEM. NMJ size is reduced in btz mutants and rescued by expressing UAS-btz with da-GAL4 or with the combination of the BG380-GAL4 and 24B-GAL4 drivers, but not with any single driver. Expressing UAS-btz RNAi with da-GAL4 or in motor neurons with BG380-GAL4, muscles with mhc-GAL4 or glia with repo-GAL4 also reduces NMJ size.
An exon junction complex-independent function of Barentsz in neuromuscular synapse growth (2021)
Figure 4

Figure 4: Btz controls NMJ size independently of the EJC. (A-H) confocal images of the NMJ on larval muscles 6 and 7 in segment A3, stained with anti-HRP (magenta) and anti-Dlg (green). (A) BG380-GAL4/+ control; (B) BG380-GAL4/+; UAS-btz RNAi/+; (C) BG380-GAL4/+; UAS-mago RNAi/+; (D) BG380-GAL4/+; UAS-eIF4AIII RNAi/+; (E) btz2/+ control; (F) btz2/Df(3R)BSC497; (G) Btz-WT; btz2/Df(3R)BSC497; (H) Btz-HD; btz2/Df(3R)BSC497. Scale bars, 30 μm (shown in A for A-D and in E for E-H). (I) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001 by unpaired t-test or Mann-Whitney test. n=15 (BG380-GAL4), n=12 (BG380>btz RNAi; BG380>mago RNAi; BG380>eIF4AIII RNAi), n=16 (btz/+), n=13 (btz/Df), or n=10 (Btz-WT; btz/Df; Btz-HD; btz/Df). Error bars show mean ± SEM. NMJ size is reduced by knocking down btz in motor neurons, but slightly increased by knocking down mago or eIF4AIII. The size deficit in btz mutants is equally well rescued by the wild type or EJC interaction-defective btz transgenes.
An exon junction complex-independent function of Barentsz in neuromuscular synapse growth (2021)
Figure 5

Figure 5: Daw signaling mediates the effects of Btz on NMJ size. (A) log2 fold changes in daw, ltl and frac mRNA in btz2/Df(3R)BSC497 larval carcasses compared to btz2/+, measured by RNA-Seq (blue) or qRT-PCR (green). **, q<0.01; *, q<0.05 by unpaired t-test corrected for false discovery rate with the Benjamini-Hochberg procedure (RNA-Seq) or **, p<0.01; *, p<0.05 by unpaired t-test (qRT-PCR). n=3 for each sample. Error bars show mean ± SD. (B-K) confocal images of the NMJ on larval muscles 6 and 7 in segment A3, stained with anti-HRP (magenta) and anti-P-Mad (green in B, C) or anti-Dlg (green in D-K). (B) btz2/+ control; (C) btz2/Df(3R)BSC497. Insets show enlargements of the boxed regions stained with anti-P-Mad. Synaptic P-Mad is lost in btz mutants. (D) UAS-daw RNAi/+ control; (E) BG380-GAL4/+; UAS-daw RNAi/+; (F) mhc-GAL4/UAS-daw RNAi; (G) UAS-daw RNAi/+; repo-GAL4/+; (H) UAS-daw/+; btz2/Df(3R)BSC497; (I) tub-GAL4/UAS-daw; btz2/Df(3R)BSC497; (J) tub-GAL4/UAS-btz; btz2/Df(3R)BSC497; (K) tub-GAL4/UAS-daw. Scale bars, 30 μm (shown in B for B-C and in D for D-K). Scale bar for insets in (B, C), 10 μm (shown in B). (L) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ns, not significant by unpaired t-test or Mann-Whitney test. n=19 (UAS-daw RNAi/+), n=9 (BG380>daw RNAi; btz/Df; tub>daw), n=23 (mhc>daw RNAi), n=10 (repo>daw RNAi; tub>daw), n=12 (btz/Df; UAS-daw), or n=14 (btz/Df; tub>btz). Error bars show mean ± SEM. Knocking down daw in motor neurons, muscles or glia reduces NMJ size. Expressing UAS-daw with tub-GAL4 rescues NMJ size in btz mutants as efficiently as expressing UAS-btz, but has no significant effect on NMJ size in a wild-type background.

Expanded View Figure Legends
Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions (2022)
Figure 3

Figure 3. Functional mapping of targeting dependencies for matrix proteins reveals multiple non-canonical Pex5 substrates.

(A) Targeting dependencies of the newly-identified peroxisomal matrix proteins were uncovered using a high-content screen. Targeting factor deletions (∆pex5 or ∆pex7) or point mutations (PEX5Y253N weakens the interaction with Pox1, a non-PTS1 protein, and PEX5N393D weakens the interaction with PTS1 proteins), were used to examine effects on peroxisomal localization of each newly-identified protein. A strain carrying a Hygromycin selection cassette in an inert locus was used as a control for no modification in peroxisomal genes.

(B) Matrix proteins targeting was dependent on the PTS1 pathway of the Pex5 targeting factor (full analysis in Dataset EV1). Presented is GFP-Yhl045w, which was not targeted to peroxisomes upon a point mutation in the PTS1-binding domain of Pex5 (PEX5N393D).

(C) The peroxisomal targeting ability of the predicted motifs was examined by fusing the last 10 amino acids of each protein to the C' of GFP, integrating the construct into an inert locus in the yeast genome, and imaging. While most motifs were unable to target GFP to peroxisomes (Appendix Fig S4B), the Yhl045w (Pxp3) motif was sufficient to target GFP to peroxisomes, in a Pex5 PTS1-binding site-dependent manner.

(D) A fluorescence anisotropy experiment demonstrated direct binding of the Pxp3 motif with purified Pex5 protein (Kd = 9.5 +/-2.9 µM), in strength similar to the binding of the known PTS1 motif from Idp3 (Kd = 5.5 +/- 0.5 µM). The plots represent the mean of three independent experiments, and the error bars represent the standard deviation.

(E) The consensus sequence of PTS1 motifs in yeast is now extended with the newly-identified, unique, residue (Tyrosine, Y) at position -2 of Pxp3.

(F) Subcellular fractionations followed by enzymatic activity assays show that untagged, native, Glr1 is active in peroxisomes, in addition to its known activity in mitochondria. Fum1 and Pox2 were used as markers for mitochondrial, and peroxisomal fractions, respectively.

(G) Glr1 C' was fused to mNeonGreen and its peroxisomal localization was analyzed by fluorescence microscopy, demonstrating that Glr1 does not rely on a free C' for targeting to peroxisomes hence it does not contain a PTS1 motif

(H) Yeast-2-hybrid assay shows Glr1 interacts in vivo with both Pex5 full-length and Pex5 N' domain.

Data information: For all micrographs, a single focal plane is shown. The scale bar is 5 µm.
Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions (2022)
Figure 5

Figure 5. Uncovering peroxisomal targeting of GID complex subunits places peroxisomes as regulators of gluconeogenesis.

(A) Gluconeogenesis utilizes unique enzymes that circumvent irreversible steps of glycolysis, one of them is the Fructose-1,6-bisphosphatase Fbp1.

(B) To ensure that gluconeogenesis does not work during glucose replete conditions, the GID complex ubiquitinates gluconeogenic enzymes shortly after glucose addition to gluconeogenic cells and marks them for proteasomal degradation. In gluconeogenic conditions, the subunit that provides substrate selectivity, Gid4, is eliminated, enabling the upregulation of necessary gluconeogenic enzymes.

(C) GFP-Gid7, a subunit of unknown function of the GID complex, is targeted to peroxisomes in gluconeogenic conditions. The images in this panel are identical to Appendix Fig S8A Glucose (logarithmic) and Glucose (stationary) images). GFP-Gid2, which was previously shown to co-localize with peroxisomes, shows a similar phenotype (Appendix Fig S8B).

(D) The peroxisomal localization of GFP-Gid7 is dependent on both PEX5 and GID2, implying that the targeting of Gid7 is mediated in the context of the complex or a sub-complex.

(E) Western blot analysis of Fbp1-HA levels in mutants Δpex3 (no peroxisomes), Δpex5 (abolished targeting of GID subunits to peroxisomes), and Δpex7 (no β-oxidation due to loss of Pot1 targeting) demonstrates that two hours after the transition from glucose to ethanol (gluconeogenic conditions), control and Δpex7 cells upregulated Fbp1-HA levels, while for Δpex3 and Δpex5 cells Fbp1-HA levels remain low. Antibodies were used against the HA tag (for Fbp1) and Actin as a loading control.

(F) Peroxisomes regulate Fbp1 levels in the transition to gluconeogenic conditions, plausibly due to partial sequestration of GID complex subunits in the peroxisomal matrix.

Data information: For all micrographs, a single focal plane is shown. The scale bar is 5 µm.
Gene expression profiling reveals insights into infant responses to group B meningococcal vaccine (2020)
Figure 5

Figure 5: Plasma proteins that differed in concentrations following vaccination in the test compare with the control vaccine group.

False-discovery rate (FDR) are displayed above and were derived from a two sample Wilcoxon rank sum test (Benjamini-Hochberg correction). Vertical lines represent the interquartile range around the median. The red dashed horizontal line is upper limit of the normal CRP reference interval (0-5mg/l). The number of individuals in each group is display in the x-axis.
Transient deSUMOylation of IRF2BP proteins controls early transcription in EGFR signaling (2020)
Figure 1

Figure 1: EGF induces rapid and transient deSUMOylation of transcriptional regulators in HeLa cells.

(A) Schematic representation of a quantitative proteome analysis that compares the SUMO proteome of serum-starved HeLa cells treated for 10 min with or without 100 nM EGF (SILAC labeling and endogenous SUMO1- and SUMO2/3-IPs).

(B) Scatterplots represent SILAC/SUMO-IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots).

(C) Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P<0.0001), as well as the three non-changing proteins SUMO2, RanGAP1 and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO-IP/immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation.

(D) Time course experiment: serum starved HeLa cells were treated with 100 nM EGF, samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.
Transient deSUMOylation of IRF2BP proteins controls early transcription in EGFR signaling (2020)
Figure 2

Figure 2: IRF2BP proteins are SUMOylated at their highly conserved C-terminus.

(A) Schematic representation of the domain structure of IRF2BP1 and IRF2BP2. The primary sequence suggests two putative SUMO-sites that are conserved among different species.

(B) Identification of the endogenous SUMO-sites in IRF2BP1 (left panel) and IRF2BP2 (right panel). HeLa cells were transfected with HA-tagged wildtype (WT) or mutant (KR) proteins, endogenous SUMO-IPs were performed and the HA-signal was analyzed by immunoblotting. Mutation of the C-terminal SUMO-site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells.

(C) Clustal omega analysis of IRF2BP1 and IRF2BP2 from H.sapiens, M.musculus, G.gallus, X.laevis, D.rerio, D.melanogaster and C.elegans shows high conservation of the C-terminal region including the SUMO-site.

(D) Schematic representation of the creation of stable, untagged and siRNA resistant IRF2BP1 WT and K579R HeLa cells. Constructs expressing IRF2BP1 variants in an pIRES-hrGFP II ("pIRES") vector were transfected, selected with antibiotics and FACS sorted for low GFP expression.

(E) Stable HeLa cells expressing pIRES-empty vector, IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non-targeting (nt) siRNA. Exogenous siRNA-resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1. * refers to an unspecific band.

(F) Wt and mutant IRF2BP1 localizes in the nucleus. After knockdown of endogenous IRF2BP1, stable IRF2BP1 (WT or K579R) cell lines were immunostained for IRF2BP1. Exogenous IRF2BP1 variants show a similar nuclear localization. Scale bar = 10 µm.

(G) IRF2BP1 wt and mutant associate with chromatin to a similar extent. HeLa cells were lysed in 0.075 % NP40 (Input). After centrifugation, the nuclei were incubated and vortexed with a nuclear extract (NE) buffer containing 170 mM NaCl. The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations, first 290 mM, then 420 mM. Wildtype IRF2BP1, the SUMO-deficient K579R mutant and the SUMOylated wildtype form (*) all behave similarly.
Transient deSUMOylation of IRF2BP proteins controls early transcription in EGFR signaling (2020)
Figure 3

Figure 3: IRF2BP1 deSUMOylation contributes to transcriptional induction of immediate early genes.

(A) Stable IRF2BP1 cell lines (knocked-down for endogenous IRF2BP1) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour. A subset of 38 EGF-dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines (at least 1.5-fold). Among them are DUSP1 (arrow), ATF3, Fos and Egr2. Each microarray was performed in triplicates, the bars show log2 values of the fold changes for wt cells (black bars) and for KR cells (grey bars). For details see Methods.

(B) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells. Gene architecture of human DUSP1. The primers at -243/-67 ("-67"), -473/-224 ("-224") and -1170/-961 ("-916") relative to the TSS were used for ChIP experiments. IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides -243 and -67.

(C) ChIP/qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between -243 and -67. Data show mean (bar) and individual data points from 2 biological replicates.

(D) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF. Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked-down for endogenous IRF2BP1, followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region -243 and -67. Data show means +/- SEM from 3 biological replicates.

(E) Q-PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1. Data show means +/- SEM from 3 biological replicates.

(F) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q-PCR (left panel), data show means +/− SEM from 5 biological replicates (left panel), and IRF2BP1 protein levels. As expected, IRF2BP1 V578A is not SUMOylated (right panel). * refers to an unspecific band.

(G) Q-PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1. Data show means +/− SEM from 4 biological replicates.
Transient deSUMOylation of IRF2BP proteins controls early transcription in EGFR signaling (2020)
Figure 4

Figure 4: IRF2BP proteins negatively regulate expression of the EGF receptor

(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1. Uba2 and beta-actin serve as independent loading controls.

(B) Model. IRF2BP1 is a SUMO-dependent transcriptional repressor of immediate early genes (IEGs). Transcription of IEGs is repressed by SUMOylated IRF2BP1, which binds to its proximal promoter. EGF receptor signaling yields at least two signals to induce IEG expression, one of which is the transient deSUMOylation of IRF2BP1.

(C) Wound healing assay: HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days, grown to 90% confluency and analysed for wound closure with or without addition of 100 ng/ml EGF. Data show means of the relative wound density +/- SEM from 4 biological replicates.

(D) Proliferation assay: HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium. One representative biological experiment is shown with means +/-SEM from 5 technical replicates.

(E) IRF2BP1, IRF2BP2, IRF2BP1+IRF2BP2 were knocked-down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis; non-targeting siRNAs were used as a control. IRF2BP1 and IRF2BP2 have distinct and overlapping functions. Experiments were done in triplicates. (F) GSEA of the highly significant enriched EGFR signaling pathway. IRF2BP1 knockdown correlates with an increase of genes involved in EGFR signalling. (G) Validation of two candidate genes: IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells, and cell lysates were analyzed by immunoblotting with the indicated antibodies.

(H) FACS-based analysis of EGFR surface expression: IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells, cells were serum starved for 16 hours, stained with fluorescent anti-EGFR antibodies and analyzed by flow cytometry. Shown are means of 3 independent experiments +/-SEM.

Expanded View Figure legends