EMBO/SourceData huggingface dataset:
Figure legend: "Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Y-maze test (F)."
NCBI gene IDs:
"Ldb2: 16826"
Figure legend: "Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Fear-conditioning test (G). Results of contextual (left) and cued (right) tests are shown."
NCBI gene IDs:
"Ldb2: 16826"
Figure legend: "Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Hot-plate test (H)."
NCBI gene IDs:
"Ldb2: 16826"
Figure legend: "Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LDB2 (A) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively."
NCBI gene IDs:
"Flag: 
HA: 
LDB2: 9079"
Figure legend: "Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LHX2 (B) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively."
NCBI gene IDs:
"Flag: 
HA: 
LDB2: 9079"
Figure legend: "Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LMO2/3/4 (C) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively."
NCBI gene IDs:
"Flag: 
HA: 
LDB2: 9079"
Figure legend: "Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates , LDB2 vs. SSBP2/3/4 (D) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively."
NCBI gene IDs:
"Flag: 
HA: 
LDB2: 9079"
Figure legend: "Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. RLIM (E). LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively."
NCBI gene IDs:
"Flag: 
HA: 
LDB2: 9079"
Figure legend: "C. Overlap of ChIP-seq peaks of LDB2 in neurosphere cells and ChIP-seq peaks of EGR1 in human cultured cell lines ECC1 and K562, at ARC"
NCBI gene IDs:
"ARC: 23237"
Figure legend: "F. In vivo tumor growth of A673 EV or GYS1-KO injected in the flanks of athymic nude mice. Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P&lt;0.0001, analyzed by two-tailed t-test."
NCBI gene IDs:
"GYS1: 2997"
Figure legend: "P. (Top) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors. (Bottom) Tumor growth of xenografts of A673 empty vector (EV) and laforin overexpression (LAOE). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P&lt;0.0001, analyzed by two-tailed t-test."
NCBI gene IDs:
"laforin: 7957"
Figure legend: "(J) Representative western blot  analysis and densitometry of zcIs13[hsp-6p::GFP] expression  in control, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E animals (one-way ANOVA with Holm-Sidak's correction, n=3 biological replicates). Data information: , bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p&lt;0.05, **p &lt; 0.01; ***p &lt; 0.001, ****p&lt;0.0001."
NCBI gene IDs:
"cest-2.2: 179917
gas-1: 181646"
Figure legend: "(M)  Immunoblot and statistical analysis of zcIs13[hsp-6p::GFP] expression  in control and nuo-6(qm200) animals fed with empty vector (C RNAi) and cest-2.2 RNAi bacteria (unpaired t-test, n=3 biological replicates). Data information: bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p&lt;0.05, **p &lt; 0.01; ***p &lt; 0.001, ****p&lt;0.0001."
NCBI gene IDs:
"cest-2.2: 179917
nuo-6: 172438"
Figure legend: "(A) Representative images of 5-day-old wt,   gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals  stained  with Oil Red O (ORO) (scale bar= full image-100 μm;  hindgut  magnification-20 μm). (B) Quantification of hindgut (left panel), foregut  (center panel) and whole body (right panel) of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2  O/E mutant  nematodes stained with  ORO (one-way ANOVA with Tukey's multiple comparison test, n=18-26 animals). (C) Quantification of mean intensity of ORO staining   in the hindgut of wt, gas-1(fc21) and gas-1(fc21); cest-2.2  O/E mutant nematodes  at different time points, starting from L4 stage (one-way ANOVA with Dunnett's post hoc correction, n=18-26 animals). (D) Mean intensity of ORO staining in the whole body  of 5-day-old wt, gas-1(fc21) and gas-1(fc21);  cest-2.2 O/E mutant nematodes  plotted against body area. Data information: , bars represent mean ±SEM points represent mean ±SEM. Across experiments, p value summary is ns= not significant, *p&lt;0.05, **p &lt; 0.01; ***p &lt; 0.001, ****p&lt;0.0001."
NCBI gene IDs:
"cest-2.2: 179917
gas-1: 181646"
Figure legend: "(E) Representative images of wt,  cest-2.2 O/E and cest-2.2 (lf) animals  stained  with  ORO. Arrows indicate mislocalized lipid droplets. (F) Quantification (mean intensity ± SEM) of  ORO staining in hindgut  (left panel), foregut (center panel) and whole body (right panel) of 5-day-old  nematodes (one-way ANOVA with Dunnett's post hoc correction, n=11-17 animals). Data information: , bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p&lt;0.05, **p &lt; 0.01; ***p &lt; 0.001, ****p&lt;0.0001."
NCBI gene IDs:
"cest-2.2: 179917"
Figure legend: "(G) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with  LipidTOX (scale bar= 100 μm). (H) Statistical analysis of whole-body LipidTOX intensity of 5-day-old nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=25-37 animals). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p&lt;0.05, **p &lt; 0.01; ***p &lt; 0.001, ****p&lt;0.0001."
NCBI gene IDs:
"cest-2.2: 179917
gas-1: 181646"
Figure legend: "(K) Representative  thin layer chromatography (TLC) plate of samples from 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes. (L) Densitometry of  TLC-separated free fatty acids (FFA) across three biological replicates in 5-day-old mutant animals. Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p&lt;0.05, **p &lt; 0.01; ***p &lt; 0.001, ****p&lt;0.0001."
NCBI gene IDs:
"cest-2.2: 179917
gas-1: 181646"
Figure legend: "(N) Sum of TLC-separated  FFA of 5-day old animals (n=5 biological replicates). (O) Quantification of individual FFA detected after  TLC  separation in 5-day-old gas-1(fc21) and  gas-1(fc21); cest-2.2 O/E mutant nematodes (n=5 biological replicates, n.d.= not detected). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p&lt;0.05, **p &lt; 0.01; ***p &lt; 0.001, ****p&lt;0.0001."
NCBI gene IDs:
"cest-2.2: 179917
gas-1: 181646"
Figure legend: "ISRE-luciferase reporter assay in 293T cells infected with BTV-8 or mock-infected (Control) and treated with 1000 U/ml of universal-IFN Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented. *"
NCBI gene IDs:
"ISRE: "
Figure legend: "GAS-luciferase reporter assay in Vero cells infected with BTV-8 or mock-infected (Control) and treated wit 5 ng/ml of IFN-γ. Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented"
NCBI gene IDs:
"GAS: "
Figure legend: "ISRE-luciferase reporter assays in 293T cells stimulated with IFN-U co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown."
NCBI gene IDs:
"ISRE: 
NS1: 
NS2: 
NS3: 
NS4: 
VP6: 
VP7: "
Figure legend: "GAS-luciferase reporter assays in Vero cells stimulated with IFN-γ co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown."
NCBI gene IDs:
"GAS: 
NS1: 
NS2: 
NS3: 
NS4: 
VP6: 
VP7: "
Figure legend: "Immunoblot of 293T cells transfected with BTV -NS2, -NS3, -NS4 (FLAG) plasmids and NiV-V (HA) and WNV-NS5 (HA) used as control for STAT1 phosphorylation inhibition and DENV-NS5 (HA) used as control for STAT2 degradation."
NCBI gene IDs:
"FLAG: 
HA: 
NS2: 
NS3: 
NS4: 
NS5: 
V: 920952"
Figure legend: "Immunoprecipitation (IP) for STAT1/2 binding using 293T transfected with BTV -NS2, -NS3, or -NS4 plasmids. NiV-V used as positive control for STAT1 and STAT2 binding, DENV-NS5 used as positive control for STAT2 binding. Cells were treated (or left untreated) with 1000 U/ml universal-IFN for the final 16 h. Immunoblots of WCE and IP were probed (IB) for STAT1, STAT2, HA (NiV V, DENV NS5), FLAG (NS2, NS3, NS4) and GAPDH."
NCBI gene IDs:
"NS2: 
NS3: 
NS4: 
NS5: 
V: 920952"
Figure legend: "STAT1 phosphorylation and (G) STAT2 nuclear translocation in BTV-NS3-transfected Vero cells detected by immunofluorescence and confocal microscopy after 30 min IFN treatment. (Scale bar = 20 µm). Arrowheads in merge images indicate examples of transfected cells."
NCBI gene IDs:
"NS3: "
Figure legend: "Immunoblots of BTV-NS3-transfected 293T cells treated with proteasome inhibitors MG or LC. Membranes were probed for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH. (B-D) Immunoblots of BTV-NS3-transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors (B) CQ or (C) NH4Cl or with increasing concentration of the (D) autophagosome formation inhibitor 3-MA, and probed (IB) for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH expression."
NCBI gene IDs:
"NS3: "
Figure legend: "293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Atg7 Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Atg7 FLAG-tagged NS3; LC3 and GAPDH."
NCBI gene IDs:
"NS3: 
Atg7: 10533"
Figure legend: "293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Beclin-1 (or siRNA control or no RNA as controls). Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Beclin-1 FLAG-tagged NS3; LC3 and GAPDH."
NCBI gene IDs:
"NS3: 
Beclin-1: 8678"
Figure legend: "Representative flow cytometry dot plots of Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and stained for BTV-VP7. Gating on VP7+ cells was used for sorting to obtain lysates from infected cells."
NCBI gene IDs:
"NS3: "
Figure legend: "Immunoblot of FACS-sorted Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and treated with IFN-U for 30min prior to fixation, staining and sorting. Membranes were probed for STAT1, P-STAT1, STAT2 and GAPDH."
NCBI gene IDs:
"NS3: "
Figure legend: "Immunofluorescence confocal images of Vero cells infected with rgBTV-8, BTV-8 ΔNS3 or mock-infected and treated with IFN-U for 30min prior to fixation and staining. Cells were stained for BTV-VP7 and (I) P-STAT1 or (J) STAT2. Nuclei were counterstained with DAPI. (Scale bar = 30 μm). (I) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV-8 ΔNS3 infected cells. (J) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV-8 ΔNS3-infected cells."
NCBI gene IDs:
"NS3: "
Figure legend: "WCE and FLAG-tag NS3 IP assay immunoblots of 293T transfected with BTV-NS3-FLAG plasmids and increasing amounts of Ubiquitin-HA plasmids, and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG) or GAPDH expression."
NCBI gene IDs:
"FLAG: 
HA: 
NS3: 
Ubiquitin: "
Figure legend: "Immunofluorescence staining of Vero cells co-transfected with BTV-NS3 and ubiquitin and acquired with Airyscan super-resolution. Scale bar = 10 μm. Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co-incidence (scale bar = 2 μm)."
NCBI gene IDs:
"NS3: 
ubiquitin: "
Figure legend: "FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected with BTV FLAG-NS3-, -NS3-K13R, -NS3-K15R, -NS3-K13/15R or -NS3-PPRY/AARH plasmid and Ubiquitin-HA plasmid and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG), STAT2 and GAPDH expression."
NCBI gene IDs:
"FLAG: 
HA: 
NS3: 
Ubiquitin: "
Figure legend: "WCE and HA-tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin-HA plasmids and increasing amounts of BTV-FLAG-NS3-K13/15R, and probed (IB) for ubiquitin (HA), BTV-NS3-K13/15R (FLAG) or GAPDH expression."
NCBI gene IDs:
"FLAG: 
HA: 
NS3: 
Ubiquitin: "
Figure legend: "ISRE-driven luciferase reporter assay in 293T cells transfected with empty expression plasmid (Control), BTV -NS2 (300 ng), -NS3 (50, 300 and 600 ng), or -NS3-K13/15R (50, 300 and 600 ng)-expressing plasmids. Mean % inductions of luciferase activity of three independent experiments are shown."
NCBI gene IDs:
"ISRE: 
NS2: 
NS3: "
Figure legend: "FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected FLAG-BTV-NS3 plasmid and HA-UB-WT or HA-UB lysine (K to R) mutants. IP were probed (IB) for ubiquitin (HA) and BTV-NS3 (FLAG) expression. WCE were probed for STAT1, STAT2, ubiquitin (HA), BTV-NS3 (FLAG), and GAPDH expression."
NCBI gene IDs:
"FLAG: 
HA: 
NS3: 
UB: "
Figure legend: "C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains. Data represent means ± SE. M: Marker; arrowhead: FIX protein; Note: the bands of FIX in F9383STOP lanes are faint with small molecular weight smears."
NCBI gene IDs:
"F9: 14071"
Figure legend: "F Measurement of blood loss over a 5-minute period after tail transection in mice at 8 weeks of age. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5."
NCBI gene IDs:
"F9: 14071"
Figure legend: "G Survival rate of mice after the tail-clip challenge. The mice were monitored for two days after tail clipping. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5."
NCBI gene IDs:
"F9: 14071"
Figure legend: "C Survival rate of mice after the tail-clip challenge. F9Y381D mice were treated with or without Cas9/sgRNA/donor DNA for 3 months. After tail-clip challenge, the survival rate of each group over 2 days was determined. Wild-type, n=9; untreated F9Y381D, n=13; DNA treated F9Y381D, n=14."
NCBI gene IDs:
"F9: 14071"
Figure legend: "D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing (Cas9/donor vector treated group) or by TA-clone sequencing (Cas9/ssODN group)."
NCBI gene IDs:
"F9: 14071"
Figure legend: "G Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level was determined in F9Y381D mice 8 weeks after HTV injection (n=5)."
NCBI gene IDs:
"F9: 14071"
Figure legend: "E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv(Cas9+G/T). (Left) HDR donor used for correction of Y381D mutation, and Illumina sequencing reads (rn) are presented. Red text indicates the correction of the F9 mutation, whereas blue text indicates the intended synonymous mutations. (Right) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA-clones (low dose) or Illumina sequencing (high dose) of treated mouseliver tissues."
NCBI gene IDs:
"F9: 14071"
Figure legend: "A: Upper panel: Strategy for the identification of oxidation resistant Ubc9 variants. Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations. Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2. For this purpose, we used a FRET-based in vitro SUMOylation assay with recombinant SUMO E1, YFP-SUMO and CFP-RanGAPtail and ATP. Lower panel: Ubc9 W103R is H2O2 resistant. Bacterial lysates containing Ubc9 wt (left panel) or Ubc9 W103R (right panel) were tested as described."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "B: Ubc9 W103 mutants are H2O2 resistant but not fully active. Left panel: Recombinant Ubc9 W103R, W103A and W103F were purified (embedded panel). Resistance against oxidation was tested under conditions of limiting E1 enzyme: 21 nM Aos1/Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP-SUMO1 and CFP-RanGAPtail. Right panel: To compare specific activities of wt and mutants in the absence of H2O2, sumoylation assays were carried out using limiting Ubc9 concentration. Reactions contained 35 nM Aos1/Uba2, 11 nM Ubc9, 85 nM each of YFP-SUMO1 and CFP-RanGAPtail and 1 mM ATP."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "B: In FRET-based assay, Ubc9 D100A is active upon H2O2 treatment. Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig. 2B."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "C: Ubc9 wt and D100A are equally active in CFP-RanGAPtail SUMOylation. Assays were with 35 nM Aos1/Uba2 and 15 nM Ubc9."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "D: Ubc9 D100A is fully competent to form a thioester with SUMO. Single turnover reactions were performed using 630 nM E1, 3,3 µM SUMO1 and 600 nM Ubc9. Reactions were stopped at different time points by addition of non-reducing buffer.."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "E: Ubc9 wt and D100A are equally active in PIAS2b - dependent SUMOylation. Assays were with 300 nM GST-p53, 170 nM E1, 120 nM Ubc9, 5 µM SUMO2 and 49 nM GST-Pias2b"
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "F: Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation. Assays were with 650 nM YFP-SP100, 110 nM of E1, 25 Ubc9, 40 nM RanBP2ΔFG and 3,5 µM SUMO1 (Left panel) or SUMO2 (Right panel)."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "A: Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT. 21 nM Aos1/Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP-SUMO1 and CFP-RanGAPtail diluted in DTT-free buffer. The final DTT concentration in the reaction was 6 µM. Prior to addition of ATP, extra amount of DTT was added at indicated final concentration."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "B: Both Uba2~Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A.100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2. One hour after H2O2 addition, 500 µM reduced glutathione was added. Samples were taken at indicated time points and the Uba2~Ubc9 disulfide was monitored by immunoblotting against Uba2. Lower panel: Quantification of the ratio between crosslinked Uba2 vs total Uba2"
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "A: Generation of polyclonal populations of stable cells. Top panel: Replacement strategy: U2OS cells transfected with pIRES-hrGFP II-based vectors were selected via addition of G418. After 4 weeks, low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks. Bottom panel: Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9: U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected. Cells were lysed in Laemmli buffer and Ubc9 and a-tubulin as loading control were detected by immunoblotting."
NCBI gene IDs:
"Ubc9: 7329
Ubc9: 22196"
Figure legend: "B: Ubc9 D100A is severely impaired for disulfide bond formation with Uba2. Stable U2OS cells were transfected with siRNA against Ubc9 for 72h, treated with 250 µM H2O2 for different time points, from 2 min to 2 hours, lysed in non-reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide (upper panel) or free Uba2 as loading control (see also Appendix Fig. S5). Lower panel: relative intensities of Ubc9 bands were measured using imageJ. Highest intensities in both cell population were set to 1."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "C: Ubc9 D100A impairs cell survival upon H2O2 treatment. Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h, transferred into 6 well plates (20 000 cells / well) and treated with increasing H2O2 concentration for 1h. Ten days later, cells were washed with PBS, fixed with 4% formaldehyde and stained with crystal violet. The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin. An arbitrary value of 1 was given to the area occupied by non-treated cells. The graph is showing the mean of 4 independent experiments. Error bars are S.E.M. *: p &lt; 0.05"
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "D: Constitutive expression of Ubc9 D100A affects cellgrowth. 103 cells were plated per 10 cm plate and cultivated for 10 days in 20% or 5% O2 incubators. Subsequently, cells were colored using crystal violet. The area of each clone was measured using ImageJ. Representative plates are shown in the upper panel. Lower left panel: Distribution of colony sizes, shown for one of the three biological replicates. Lower right panel: Comparison of the colonies size between \"Ubc9 wt\"and \"Ubc9 DA\" cells. The average size of \"Ubc9 wt\" was set to 1. Shown is the mean of 3 independent experiments, each of which was performed in technical triplicates. Error bars are S.E.M."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "A: Phosphorylation of ATM is impaired in Ubc9 D100A cells. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "B: Phosphorylation of gamma-H2AX is impaired in Ubc9 D100A. Experiment was performed as in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies."
NCBI gene IDs:
"Ubc9: 22196"
Figure legend: "C: Phosphorylation of Chk2 is impaired in Ubc9 D100A. Experiment was performed as described in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies."
NCBI gene IDs:
"Ubc9: 22196"