EMBO/soda-nel huggingface dataset:
Absence of RNA binding protein FXR2P prevents prolonged phase of kainate-induced seizures (2021)
Figure 2

Figure 2. Kainic acid- and pilocarpine-induced epileptic seizures in WT and Fxr2 KO mice.

A The different stages of seizures based on increased severity.

B Average seizure score of KA-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h. * p < 0.05 compared to WT mice (two-way Repeated Measures ANOVA; interaction effect of time and genotype; F1,666 = 10.05, p = 0.0016). KA-treated WT mice (n=16), KA-treated Fxr2 KO mice (n=12). Data are presented as mean ± Standard Error of the Mean (SEM).

C Responsiveness to KA of WT (black, n=16) and Fxr2 KO mice (magenta, n=12). *** p < 0.001 compared to Fxr2 KO mice (two-tailed Student's t-test; t26 = 5.72, p < 0.001). Data are presented as mean ± SEM.

D Average seizure score of pilocarpine-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h (two-way Repeated Measures ANOVA). Pilocarpine-treated WT mice (n=6), pilocarpine-treated Fxr2 KO mice (n=6). Data are presented as mean ± SEM.

E Responsiveness to pilocarpine (PC) of WT (n=6) and Fxr2 KO mice (n=6). (two-tailed Student's t-test). Data are presented as mean ± SEM.
Absence of RNA binding protein FXR2P prevents prolonged phase of kainate-induced seizures (2021)
Figure 3

Figure 3. Identification of FXR2P target mRNAs.

A Schematic representation of the experimental strategy for the RIP-seq pipeline. Representative Western blot showing the FXR2P pull-down. FXR2P was precipitated from WT and Fxr2 KO mice brain extracts using the 1G2 antibody (Fernandez et al, 2015) and analyzed by Western blotting. Input, 10%. PSD-95 mRNA was used as control to verify the specificity of the RIP-seq.

B PSD-95 mRNA had lower read counts in Fxr2 KO samples (n=3) compared to WT samples (n=3) (+ adjusted p < 0.1, two-tailed Student's t-test). Data are presented as mean ± SEM.

C Enrichment analysis of the 14'809 transcripts shows 488 genes (red) that pass the FDR threshold (adjusted p-value cut-off at 0.1).

D Left, gene ontology of the 488 FXR2P candidate mRNAs across the top 10 cellular components. Right, biological processes specific for glutamate are highlighted. -Log10 q-values were normalized according to the maximum q-value in the list of gene ontologies. The red dashed line indicates the threshold for significant enrichment in these gene ontologies.

E Left, FXR2P binding motif sequence as described by Ray et al (2013). Right, distribution of match scores between FXR2P target and non-target mRNAs. FXR2P target mRNAs show on average higher match scores than FXR2P non-target mRNAs (Kolmogorov-Smirnov test, p < 0.001).
Absence of RNA binding protein FXR2P prevents prolonged phase of kainate-induced seizures (2021)
Figure 5

Figure 5. Validation of glutamatergic FXR2P mRNA targets.

A Representative Western blot and quantification of GluK5, PSD-95, GluN2B and mGluR5 proteins. Endogenous GluK5, PSD-95, GluN2B and mGluR5 in Fxr2 KO mice (n=9) compared to WT mice (n=11). * p < 0.05, ** p < 0.01, significant difference between WT and Fxr2 KO (two-tailed Student's t-test: GluK5: t18 = 0.87, p = 0.394; PSD-95: t18 = 2.29, p < 0.05; GluN2B: t18 = 3.09, p < 0.01; mGluR5: t18 = 3.74, p < 0.01). Quantified bands are highlighted with an asterisk or squared bracket. Data are presented as mean ± SEM.

B Bar plot showing the mRNA levels of Grin2b, Grm5 and Dlg4, normalized for the average of Vinculin and Gusb mRNA levels in WT (n=5) and Fxr2 KO (n=5) hippocampi (two-tailed Student's t-test). Data are presented as mean ± SEM.
Absence of RNA binding protein FXR2P prevents prolonged phase of kainate-induced seizures (2021)
Figure 6

Figure 6. ERK-mediated signaling in male WT and Fxr2 KO.

A Bar plot showing phospho-ERK1/2 over total ERK1/2 levels in vehicle- and KA-treated WT (closed and open black circles, vehicle-treated WT n=6; KA-treated WT n=6) and Fxr2 KO mice (closed and open magenta circles, vehicle-treated Fxr2 KO n=5; KA-treated Fxr2 KO n=6). *** p < 0.001 comparison between vehicle- and KA-treated WT (two-way ANOVA: interaction effect between genotype and treatment: F1,19 = 24.66, p < 0.001). Data are presented as mean ± SEM.

B Phospho-ERK1/2 levels in vehicle- and pilocarpine-treated WT (n=4-6) and Fxr2 KO mice (n=4-6). Both genotypes responded with long-lasting seizures upon pilocarpine administration * p < 0.05 (two-way ANOVA: main effect for treatment: F1,16 = 10.98, p < 0.01). Data are presented as mean ± SEM.

C Phospho-ERK1/2 levels in control and KA-treated WT (both n=6) and Fxr2 KO mice (both n=6) sacrificed at different time intervals [0, 20, 40 and 60 min]. * adjusted p < 0.05, ** adjusted p < 0.01 (two-way ANOVA: main effect for genotype: F1,40 = 5.99, p < 0.05; interaction effect genotype and time; F3,40 = 4.11, p = 0.012; p < 0.05 and p < 0.01 with Bonferroni correction for 40 and 60 min, respectively). Data are presented as mean ± SEM.
SUMOylation promotes protective responses to DNA-protein crosslinks (2019)
Figure 1

Figure 1.

Formaldehyde triggers a dynamic chromatin SUMOylation response

Chromatin-enriched fractions of HeLa cells exposed to the indicated genotoxic agents for 1 h were subjected to immunoblot analysis using SUMO2/3 antibody.

As in (A), except that cells were preextracted in 0.2% Triton X-100, fixed and immunostained with SUMO2/3 antibody. Representative images are shown. Scale bar, 5 μm.

Relative DPC levels in cells treated as in (A) were quantified using a KCl/SDS precipitation assay (mean±SD; n=3 independent experiments).

HeLa cells were treated with formaldehyde for 1 h, and where indicated propagated for an additional h in the absence of formaldehyde (recovery). Cells were then fractionated into soluble and chromatin-enriched fractions and immunoblotted with SUMO2/3 and SUMO1 antibodies.

As in (D), but using HeLa cells transfected with non-targeting control (CTRL) or SPRTN siRNAs.

Experimental set-up for global proteomic analysis of formaldehyde-induced SUMOylation changes.

Mass spectrometry-based analysis of formaldehyde-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde as shown in (F) were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment (Table EV1) were subjected to network analysis using the STRING database, at the default interaction confidence setting of 0.4. Proteins not connected to the network were omitted.

SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified as in (G) and immunoblotted with indicated antibodies.

SUMO target proteins displaying at least 2.5-fold upregulation of SUMOylation after formaldehyde treatment were mapped to the human proteome, which was annotated with Gene Ontology (GO) terms. Enrichment analysis was performed to find terms significantly enriched for formaldehyde-induced SUMOylated proteins, using Fisher Exact testing with multiple-hypothesis correction to achieve a final q-value of <0.02. A relative score was derived from a combination of the enrichment ratio and the q-value. A full list of all enriched terms is available (Table EV2). GOBP, GO Biological Processes; GOCC, GO Cellular Compartments; GOMF, GO Molecular Functions.
SUMOylation promotes protective responses to DNA-protein crosslinks (2019)
Figure 2

Figure 2.

DPCs are direct targets of SUMO-dependent modification

Principle of 5-azadC-induced DNMT1 DPC formation. See main text for details.

HeLa cells were transfected with non-targeting control (CTRL) or DNMT1 siRNAs, exposed or not to 5-azadC and collected 2 h later. Chromatin-enriched fractions were immunoblotted with antibodies to SUMO2/3, DNMT1 and MCM6 (loading control).

Representative images of HeLa cells that were treated as in (B) and co-immunostained with antibodies to DNMT1, SUMO2/3 and PCNA. Scale bar, 10 μm.

Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with indicated siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).

Extracts of HeLa/GFP-DNMT1 cells treated with 5-azadC for the indicated times were subjected to GFP immunoprecipitation (IP) under denaturing conditions followed by immunoblotting with SUMO2/3 and GFP antibodies.

HeLa cells were subjected to consecutive rounds of transfection with DNMT1 siRNA targeting the UTR and expression plasmid encoding WT or catalytically inactive (CI) GFP-DNMT1. Cells were then left untreated or incubated with 5-azadC for 2 h, and SUMOylation of GFP-DNMT1 was analyzed as in (E). Asterisk denotes unmodified GFP-DNMT1 recognized by the SUMO2/3 antibody due to weak cross-reactivity.

Experimental set-up for global proteomic analysis of 5-azadC-induced SUMOylation changes.

Mass spectrometry analysis of 5-azadC-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to 5-azadC as shown in (G) were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. Proteins displaying significantly altered SUMOylation in response to 5-azadC treatment relative to the control condition were visualized through two-sample t-testing using permutation-based FDR to achieve q-values of <0.05 (Table EV3).

As in (H), except that SUMO target proteins displaying altered SUMOylation upon DNMT1 knockdown are shown. Both control (CTRL) and DNMT1 siRNA-transfected cells were treated with 5-azadC.
SUMOylation promotes protective responses to DNA-protein crosslinks (2019)
Figure 3

Figure 3.

SUMOylation promotes resolution of DNMT1-DNA adducts and cell fitness upon 5-azadC treatment

Chromatin-enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5-azadC for 2 h were analyzed by immunoblotting with SUMO2/3 and Actin antibodies.

HeLa cells stably expressing GFP-DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5-azadC for 2 h. Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal. Mean detergent-resistant GFP signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown. Representative images are shown in Fig EV2C.

Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).

As in (B), except that untransfected HeLa cells were exposed or not to 5-azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor (SUMO-E1i).

HeLa cells treated with 5-azadC for 2 h in the presence or absence of SUMO-E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal, and immunostained with DNMT1 antibody. Mean detergent-resistant DNMT1 signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.

Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5-azadC doses for 2 h was assayed as in (C) (mean±SEM; n=3 independent experiments; **p<0.01, n.s. not significant, Student's t-test).

iPOND analysis of 5-azadC-induced DNMT1 trapping and SUMOylation. HeLa cells were mock-treated or pre-incubated with 5-azadC for 5 min before addition of EdU for 10 min. Cells were then washed and incubated with thymidine for 0 (pulse) or 60 min (chase) before performing iPOND.

HeLa cells stably expressing GFP-DNMT1 were pre-incubated or not with SUMO-E1i for 30 min, and where indicated 5-azadC was added to the medium for an additional 60 min. Cells were then processed for GFP immunoprecipitation (IP) followed by immunoblotting for the indicated proteins.

Immunoblot analysis of chromatin-enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block, pulse-labeled with 5-azadC for 30 min and grown in the presence or absence of proteasome inhibitor (MG132) for the indicated times.

As in (I), except that cells were treated or not with aphidicolin following pulse-labeling with 5-azadC.
SUMOylation promotes protective responses to DNA-protein crosslinks (2019)
Figure 4

Figure 4.

The SprT protease ACRC but not SPRTN is recruited to DPCs in a SUMO/SIM-dependent manner

Domain structure of the human SprT proteases SPRTN and ACRC, showing location of SprT protease domains and additional motifs, including a ubiquitin-binding UBZ domain in SPRTN and an N-terminal cluster of SIMs (sequence highlighted) in ACRC. Alanine substitutions introduced to disrupt the functionality of the ACRC SIMs (SIM*) are indicated.

Representative images of U2OS cells stably expressing GFP-SPRTN or GFP-ACRC that were transfected with control (CTRL) or UBC9 siRNAs, exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme (UBA1) inhibitor TAK-243 (Ub-E1i) as indicated and fixed one h later. Scale bar, 10 μm.

Quantification of data in (B), showing proportion of U2OS/GFP-SPRTN cells displaying nuclear GFP-SPRTN foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).

As in (C), but showing proportion of U2OS/GFP-ACRC cells displaying nuclear GFP-ACRC foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).

U2OS cells expressing GFP-ACRC were exposed to the indicated genotoxic agents (formaldehyde: 600 μM, 1 h; UV: 20 J/m2, 6 h recovery; hydroxyurea (HU): 2mM, 24 h; mitomycin C (MMC): 40 ng/mL, 24 h), preextracted and fixed, and analyzed by microscopy. Representative images are shown. Scale bar, 10 μm.

HeLa cells transfected with plasmids encoding GFP alone or indicated GFP-ACRC alleles were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO22-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.

Representative images of U2OS cells expressing indicated GFP-ACRC alleles that were treated with 5-azadC in the presence or absence of SUMO inhibitor (SUMO-E1i), fixed 2 h later and immunostained with DNMT1 antibody. Scale bar, 10 μm.

Quantification of data in (G), showing proportion of cells displaying GFP-ACRC co-localization with DNMT1 foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).
SUMOylation promotes protective responses to DNA-protein crosslinks (2019)
Figure 5

Figure 5.

The ACRC ortholog GCNA-1 and SUMOylation protect against DPC toxicity in C. elegans

Domain structure of human ACRC and its C. elegans ortholog GCNA-1. The GCNA-1 deletion (del) introduces a frameshift at E364, giving rise to a truncated protein containing an aberrant 22-residue C-terminal addition.

HeLa cells transfected with plasmids encoding GFP alone, GFP-ACRC or GFP-tagged C. elegans GCNA-1 were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO23-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.

Schematic representation of the C. elegans gcna-1 locus, depicting mutants generated. Loss-of-function gcna-1 allele (emcSi31) was created by knock-in of a gfp selection cassette (GFP-SEC) in the start codon (see Fig EV4A). gcna-1 deletion (emc52), leading to a frameshift and premature stop codon in the coding sequence, and E364Q point mutant (emc53) alleles were generated by CRISPR/Cas9 targeting of exon 6.

GFP expression driven by the gcna-1 promoter was observed in germ cells, proliferating embryos and young larvae but not in post-mitotic tissues in the head (see also Fig EV4B). Scale bars, 50 µm.

Formaldehyde survival of wild type (wt), dvc-1, gcna-1 loss-of-function (lof) and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=4 independent experiments).

Cisplatin survival of wild type, dvc-1, gcna-1 and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=3 independent experiments).

Formaldehyde survival of gcna-1 deletion (del) and E364Q mutant C. elegans (mean±SEM; n=2 independent experiments).

Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).

Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or gei-17 RNAi bacteria (mean±SEM; n=2 independent experiments).

Formaldehyde survival of wild type and gcna-1 deletion (del) mutant C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).

Expanded view figure legends
Local activation of mammalian separase in interphase promotes double strand break repair and prevents oncogenic transformation (2018)
Figure 1

Figure 1. Human separase localizes to DSBs in post-replicative cells.
(A) Following transfection of given siRNAs and synchronization in G2-phase, Hek293 cells were DRB- or mock-treated and then subjected to IFM using the indicated antibodies. Lower panels display a 3-fold magnification of the boxed area shown above. Scales bars correspond to 5 and 1 μm, respectively. See figure EV1D for corresponding immunoblot.

(B) Hek293 cells were thymidine-arrested for 20h, mock- or OHT-treated to induce DSBs by nuclear accumulation of ER-AsiSI and then subjected to ChIP-multiplex PCR.

(C) ChIP samples from (B) were analysed by qPCR. Shown are averages (bars) of 3 independent experiments (dots).

(D, E) Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed by IP-Western (D) and by IFM for γH2AX- and Myc-separase-positive foci (see Figure EV2E). The quantification of the IFM in (E) shows averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each.
Local activation of mammalian separase in interphase promotes double strand break repair and prevents oncogenic transformation (2018)
Figure 2

Figure 2. Separase supports HDR but not NHEJ
(A-C) Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected to immunoblotting (A) and flow cytometry to quantify the percentage of GFP positive cells (B) and PI-stained cellular DNA (C). The GFP quantification in (B) displays averages (bars) of 3 independent experiments (dots).

(D, E) Co-depletion of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed by GFP-flow cytometry (D) and immunoblotting (E). Shown in (D) are averages (bars) of 2-3 independent experiments (dots).
Local activation of mammalian separase in interphase promotes double strand break repair and prevents oncogenic transformation (2018)
Figure 3

Figure 3. Human separase locally cleaves Rad21 at DSBs.
(A) Transgenic Hek293 cells constitutively expressing a separase sensor (cartoon below) and inducibly expressing Myc-separase were transiently transfected to express Flag-AsiSI-ER, Dox- and/or OHT-treated in G2-phase and analysed by (IP-)Western.

(B) Cells from (A) were analysed by IP-Western for cleavage of endogenous Rad21. As a control, in vitro-translated (IVT) Rad21 was incubated with hyperactive separase-SA or a protease-dead (PD) variant (Boos et al, 2008).

(C) Sensor expressing cells were treated in G2-phase with DRB and nocodazole for 6 and 2 h, respectively, prior to chromosome spreading and IFM using Hec1- and γH2AX-antibodies. The separase sensor was detected based on autofluorescence of eGFP and mCherry, while Hec1- and γH2AX-antibodies were detected with corresponding Cy5- and marina blue-labeled secondary antibodies, respectively. Note that sizes of spread chromosomes vary greatly with buffer conditions, which is why no scale bar is shown.
Local activation of mammalian separase in interphase promotes double strand break repair and prevents oncogenic transformation (2018)
Figure 4

Figure 4. NES phosphorylation and RG-repeat methylation of separase in response to DSBs
(A-C) Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated. (A and B) Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst others, a pan-specific antibody against phosphorylated serine (A, pan-pS) or a separase antibody specific for phosphorylated Ser1660 (B, pS1660). (C) DNA damage-induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition. G2-enriched Hek293 cells were treated with KU-55933 (0.3 μM) and/or DRB and analyzed by (IP-)Western 12 h thereafter using the indicated antibodies.

(D) Preventing NES phosphorylation spoils nuclear localization of separase in response to DSBs. HeLaK cells expressing N-terminally NLS-eGFP-tagged separase variants were treated with DRB or carrier solvent (− DRB) for 4 h and then subjected to IFM using anti-γH2AX and anti-Nup153 to visualize sites of DNA damage and nuclear pore complexes, respectively. Transgenic separase was detected based on the eGFP autofluorescence. Note that due to their relatively high nuclear concentration co-localization of separase-WT and -S1660D with γH2AX-foci is not discernable. Scale bar = 5 μm.

(E) In vitro phosphorylation of separase on Ser1660 by ATM kinase. Incubation of GST-p53 (amino acids 9-22), GST, separase-WT, or separase-S1660A with recombinant ATM-WT, ATM-KD (kinase dead), and/or KU-55933 in presence of [γ-33P]-ATP was followed by immunoblotting and autoradiography.

(F) Arg-methylation of RG-repeats mediates recruitment of separase to DSB-containing chromatin. Myc-separase-WT or -KG expressing cells were treated with DRB as indicated and analysed by (IP-)Western and Coomassie staining.

(G) In vitro Arg-methylation of separase's RG-repeats by PRMT1. Incubation of histone H4, separase-WT, or separase-KG with recombinant PRMT1 or reference buffer in presence of S-adenosyl-L-[methyl-3H]-methionine was followed by Coomassie staining and autoradiography.
Local activation of mammalian separase in interphase promotes double strand break repair and prevents oncogenic transformation (2018)
Figure 5

Figure 5. NES-phosphorylation and Arg-methylation of separase is required for proper DSB-repair.
(A) Persistence of γH2AX- and MDC1-positive foci in absence of NES-phosphorylation or RG-methylation of separase. Transfected Hek293T cells were siRNA- and Dox-treated to deplete endogenous separase and induce expression of the indicated, siRNA resistant Myc-tagged separase variants, respectively. Then, they were constitutively (− washout) or transiently exposed to DRB (+ washout) and finally quantitatively assessed by IFM for γH2AX- and MDC1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm.

(B) Schematic of the experiment.

(C) Recruitment of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG to DSBs. Cells from (A) were treated as indicated and analysed by IFM for co-localization of Myc-separase with γH2AX-foci. Scale bar = 5 μm.

(D) Interaction of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG with γH2AX. Cells from (A) were subjected to (IP-)Western analysis as indicated. Lanes that illustrate cellular levels of cyclin A2 and γH2AX after DRB-washout and those that analyse interaction of Myc-separase variants with γH2AX in presence of DRB are labeled by red arrows and blue arrow heads, respectively.