EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) IF of CBX2 and SUMO2/3 in PN0 (n=10) and PN5 (n=12) zygotes. Data information: Scale bars, 10µm. (B) ChIP-qPCR analyses of HEK2935xUAS cells co-expressing GAL4DBDCBX2.1 either with DAXXMycHis or DAXXΔSIM1/2MycHis. Data are means ± SEM (n = 3). (C) IF of EGFP and DAXX in Daxxm-z+ PN5 zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA (100ng/µl). Data information: Scale bars, 10µm. (D) Left: Boxplot displaying EGFP signal intensities at PCH relative to euchromatin in Daxxm-z+ zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA. Right: Boxplot displaying signal intensity of EGFP at euchromatin in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, N.S.: no statistical difference, P>0.05, t-test. (E) Sequence alignment of mouse and human CBX2 proteins with SUMO sites indicated. (F) Ni-pulldown of 6xHisSUMO1/2/3-conjugated proteins from HEK293 extracts expressing 6xHis-tagged SUMO1, SUMO2 or SUMO3 in combination with CBX2-EGFP. CBX2 and SUMO-conjugated CBX2 were detected with an anti-GFP antibody. (G) Ni-pulldown of 6xHis-SUMO2 conjugated proteins from HEK293 extracts transiently co-expressing 6xHis-SUMO2 with CBX2-EGFP or CBX24KR-EGFP. Detection of CBX2 (H) IF of DAXX and EGFP in wild-type PN5 zygotes, micro-injected with Cbx2 or Cbx24KR-EGFP mRNA (100ng/µl). Data information: Scale bars, 10µm. (I) Left: Boxplot displaying signal intensity of DAXX at PCH relative to mean intensity at pat-PCH in wild-type zygotes micro-injected with Cbx2 or Cbx24KR-EGFP mRNA. Right: Absolute intensities of EGFP in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. ***P<0.001, t-test.
Figure 3A Representative images of neurons stained with the neuron specific anti-NCAM antibody, ROS sensible fluorescent probe DCF, and the nuclei dye Hoechst. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM positive controls- and PKAN humanneurons, infected or not with Ngn2-PANK2-LV. Data presented as means + SEM of at least three independent experiments (One-way ANOVA). B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1. Scale bar 20 μm. ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots. Data presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).
Figure 4A. Single-cell histories of OCT4 dynamics were classified into 3 time periods. Maternal OCT4 encompasses the levels throughout the lifetime of the mother cell. Inherited OCT4 represents the levels established within 1 h of division. Autonomous OCT4 refers to the OCT4 dynamics during the remaining lifetime of the daughter cell. B. OCT4 mixing as a function of cell history. 30 randomly chosen cells were colored according to their OCT4 expression level at 8 h after division. C. Variance in final OCT4 expression explained over time during maternal, inherited, and autonomous periods of cell history. At each time point, the coefficient of determination (R2) between total OCT4 expression levels and final OCT4 expression at 8 h was calculated. D. Estimate of variance explained by asymmetric distribution of OCT4 to daughter cells. The same R2 over time mother-daughter traces were randomly sampled from a set of mother-daughter traces that contained sister-cell pairs (blue line) or only one randomly selected sister cell from each pair (red line). E. Difference in variance explained between red and blue traces in panel D. The black line represents the actual difference; the red line shows a smoothing (moving average = 1.25 h) of the black trace to highlight the overall trend.
Figure 3(A) Schema of in-crosses used to generate clutches for survival assay. (B) The predicted mendelian (1:2:1) and experimental ratio of genotypes of progeny from smyhc1R673H/+ in-crosses demonstrates survival at the indicated ages. This ratio quickly skewed toward an overrepresentation of smyhc1+/+ fish with no representation of smyhc1R673H/R673H after 5 dpf. There is also dropout of smyhc1+/R673H fish into adulthood. Number of fish counted per group is indicated.
Figure 7A, B CD4+T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). CBL (A) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments. A, B CD4+T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). PI3KR1 (PI3Kp85α) (B) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.
Starvation-Induced BimT116 Phosphorylation Disrupts the Bim-Beclin- 1 Interaction.Figure 5(A) Bim(EL) EE/empty vector (IP negative control) or Bim(EL) EE/Beclin 1-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of cells with Bim(EL) EE/Beclin 1-Flag were starved in HBSS for 2 hr. Anti-Flag antibody (M2) was used for immunoprecipitation, and blots were probed as indicated. Data are shown as mean ± SD. ∗∗∗p < 0.001. (B) Panel i: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were starved in HBSS for 2 hr. Blots were probed as indicated. LC3-II/tubulin ratio of siRNA-transfected cells is set as 1 (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant. Panel ii: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were treated with Baf for 2 hr; one set of transfections were starved and treated with Baf for 2 hr. Blots were probed as indicated and analyzed as in (Bi). ∗∗p < 0.01; NS, not significant. (C) HeLa cells were transfected with control plasmid /GFP-LC3 (3:1), or Bim(EL) EE/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. NS, not significant. (D) BimS-Beclin 1 binding is not sensitive to HBSS starvation. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag (two replicates), BimL-EE (L)/Beclin 1-Flag (two replicates) or BimS-EE (S)/Beclin 1-Flag (two replicates) were transfected into HeLa cells. After 20 hr, one of EL-Beclin 1-Flag transfections, one of L-Beclin-Flag transfections and one of S-Beclin 1-Flag transfections were subjected to 2 hr HBSS starvation. Anti-Flag (M2) was used for immunoprecipitation. (E) HBSS starvation increases Bim T116 phosphorylation. Bim(EL) EE or BimEE(EL)-T116A were transfected into HeLa cells. After 20 hr, one set of Bim(EL) EE transfections and one of Bim(EL) EE-T116A were starved for 2 hr in HBSS. Blots were probed with the indicated antibodies. (F) Bim T116 phosphorylation disables Bim-Beclin 1 interaction. Bim(EL) EE /vector (IP negative control), Bim(EL) EE/Beclin 1-Flag (in duplicate), or Bim(EL) EE-T116E (phospho-mimic, designated as T116E here)/Beclin-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of Bim(EL) EE/Beclin 1-Flag transfections and one of Bim(EL) EE-T116E/Beclin 1-Flag transfections were starved for 2 hr in HBSS (Starv). Anti-Flag (M2) was used for immunoprecipitation. (G) HeLa cells were transfected with control plasmid /GFP-LC3, Bim(EL)-EE/GFP-LC3 (3:1), or Bim(EL)-EE T116E/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and the cells were then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.
Fig.Figure 5Metabolomics profile of GC muscle of 4-month-old HSA-Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of compounds Fold change of phospholipid precursors in GC muscles of 4-month-old HSA-Cre and control mice measured by liquid-chromatography (LC) tandem mass spectrometry (MS/MS) (n=7-4, *P<0.05). Fold change of Lactate/Pyruvate, Pyruvate/AcetylCoA, Pyruvate/Citrate, ATP/AMP, and ATP/ADP ratios in GC muscles of 4 month-old HSA-Cre and control mice (n=7-4, *P<0.05).
Figure 3A, Immunostaining of cytokeratin 8 (CK8), a marker of epithelial cell lineage, showed that epithelial alignment is collapsed and the trophoblast invades the stroma at implantation sites in Rb1f/f mice, while epithelial alignment is persistent and trophoblast invasion into the stroma is compromised in Rb1d/d mice. Scale bar = 200μm; arrowhead, an embryo. B, Transmission electron microscopic analyses (TEM) showed that the uterine epithelium surrounding the embryo disappears and the trophoblast contacts directly with the stroma at 2000h on day 5 in Rb1f/f mice, while the uterine epithelium is persistent and the embryo does not attach to the stroma in Rb1d/d mice. Scale bar = 10µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast. C, TEM showed that fragmented luminal epithelial cells with cytoplasmic lipid droplets are engulfed by the trophoblast in the control mice (Rb1f/f mice). Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; yellow dotted line circle, cytoplasmic lipid droplets in the luminal epithelium; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast. D, TEM showed that the microvilli, markers of uterine receptivity, were observed at the surface of intact luminal epithelium on day 4 of pregnancy and epithelial cells with cytoplasmic lipid droplets were observed at 2000h on day 5 of pregnancy in the control mice (Rb1f/f mice). Scale bar = 2µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast. E, Immunofluorescence of MFG-E8, a marker of programmed cell death, showed that MFG-E8 is not expressed at the implantation sites of Rb1f/f mice at 0900h on day 5 and Rb1d/d mice at 0900h and 2000h on day 5, but is expressed at the implantation sites of Rb1f/f mice at 2000h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; arrowhead, embryo.
Figure 6A-C. Nucleosome occupancy and condensin binding during mitosis upon RSC loss of function. Fission yeast cells expressing Cnd2-GFP were arrested in early mitosis, mitotic indexes determined (A), and cells processed for ChIP against H3 and H4 (B). Averages and s.d. calculated from 3 ChIPs per antigen, each performed on 3 biological replicates. Values for prl53 and ecm33 were multiplied by 4 to facilitate reading. A-C. Nucleosome occupancy and condensin binding during mitosis upon RSC loss of function. Fission yeast cells expressing Cnd2-GFP were arrested in early mitosis, and cells processed for ChIP against Cnd2-GFP (C). Averages and s.d. calculated from 3 ChIPs per antigen, each performed on 3 biological replicates. Values for prl53 and ecm33 were multiplied by 4 to facilitate reading. D. Cnd2-GFP levels by western blotting. E-F. RSC localization at condensin binding sites during mitosis. Mitotically-arrested cells were subjected to ChIP against Snf21-FLAG. Averages and s.d. calculated from 12 ChIPs on 6 biological replicates are indicated. Values for 5S and gly05 were multiplied by 3. *** P<0.001, ** P<0.01, ° P>0.05. High-occup. and Low-occup. refer to condensin high-occupancy and low-occupancy binding sites, respectively.
Figure 2A ORO staining of SVF cells isolated from iWAT of male mice. Differentiation was induced with curcumin up to day 8. C ORO staining of 3T3-L1 cells. Differentiation was induced with curcumin up to day 8. B mRNA expression of adipogenesis-related factors of SVF cells isolated from iWAT of male mice. D mRNA expression of PPARγ, C/EBPα and FABP4 expression of 3T3-L1 cells treated with curcumin. E LC-MS/MS quantification of the m6A/A in mRNA of iWAT of mice fed with HFD and HFD+CUR groups. mRNA expression levels of m6A regulator in iWAT from mice in HFD and HFD+CUR groups. protein expression levels of m6A regulator in iWAT from mice in HFD and HFD+CUR groups. protein expression levels of m6A regulator in 3T3-L1 cells were analyzed H LC-MS/MS quantification of the m6A/A in mRNA of 3T3-L1 cells. mRNA expression levels of m6A regulator in 3T3-L1 cells were analyzed by qPCR. K Oil Red O staining of Vector or OE-ALKBH5 transfected 3T3-L1 cells. Differentiation was induced with curcumin up to day 8. L mRNA expression of PPARγ, C/EBPα and FABP4 expression of cells
Figure 6 Figure 6. MGC formation is dependent on iNOS induced DNA damage A BMMΦ from wt and iNOS-/- mice were stimulated with LP for 6 days and quantified for MGC formation by hemacolor staining. Depicted are means ± SEM of 3 independent experiments. B BMMΦ were stimulated with LP and aminoguanidine (0.1 - 1mM) for 6 days and quantified for MGC formation as in (A). Depicted are means ± SEM of 4 independent experiments. C BMMΦ from wt and iNOS-/- mice were stimulated with LP or supplemented with SNP (300µM) for 6 days and quantified for MGC formation as in (A). Depicted are means ± SEM of 3 independent experiments. D, E BMMΦ from wt and iNOS-/- mice were stimulated with LP and aminoguanidine (1mM) for 6 days and lysed with RIPA-buffer for western blot analysis of γH2Ax (D) D, E BMMΦ from wt and iNOS-/- mice were stimulated with LP and aminoguanidine (1mM) for 6 days and lysed with RIPA-buffer for western blot analysis of p53 levels (E). F Representative images of mouse ears8 weeks p.i. with PBS+ 5% mineral oil only (vehicle) or with addition of 105 CFUs BCG-RFP. Infected area was stained for immunofluorescence with nitrotyrosine (green) and DAPI (blue) and counterstained with hematoxylin/eosin; scale bar, 100µm. G Whole ear lysates from 3 and 8 weeks of BCG-RFP infected wt and iNOS-/- mice were plated on 7H11 agar and quantified for CFUs after 3 weeks. Depicted are measn ± SEM of 4 mice. H Surgically dissected ears from wt and iNOS-/- mice were frozen as cryo-blocks, cut and stained by hematoxylin/eosin for analysis by microscopy. Depicted are means ± SEM of 3-7 mice. I BMMΦ from wt and p47-/- mice were stimulated with LP for 6 days and quantified for MGC formation as in (A). Depicted are means ± SEM of 3 independent experiments.  J BMMΦ were stimulated with LP and diphenyleneiodonium (DPI) (0.25 - 0.5µM) for 6 days and quantified for MGC formation as in (A). Depicted are means ± SEM of 3 independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001 (two-tailed unpaired t-test).
Figure 3A Sensitivity to 6-AU of the indicated cells expressing chromosomally integrated ULP2-Myc derivatives. Assays were performed The schematic diagram below shows full-length Ulp2 with a 9Myc epitope tag and the segments defining the N- and C-terminal domains and the catalytic ULP domain (UD). B Immunoblot assay of the Ulp2-Myc derivatives expressed in the strains C ChIP analysis of Flag-Ulp2 derivatives performed with the same PCR probes W303a cells carrying pRS424-GAL1 plasmids that expressed N-terminally Flag-tagged Ulp2 derivatives-specifically, full-length, NTD (1-403) and CTD (667-1034)-were grown to mid-log phase in SD-Trp medium containing 2% galactose. Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT, full length of Ulp2 (*, P <0.05). D Co-IP assay for interaction between Ulp2-Myc derivatives and Flag-H2B in strains transformed with a pRS314 plasmid expressing Flag-tagged H2B. Immunoprecipitated (IP) proteins from anti-Myc agarose beads were analyzed by immunoblotting (IB) using anti-Myc or anti-Flag antibodies. The upper and middle panels show anti-Myc blotting and anti-Flag blotting, respectively. The levels of bound H2B proteins were measured relative to INPUT H2B levels shown in the lower panel. E Chromatin association assays using strains After fractionation into chromatin (pellet) and soluble (supernatant) components, fractions were analyzed by immunoblotting using the indicated antibodies. F Quantitation of chromatin association of Ulp2-Myc derivatives in (E). The levels of Ulp2 protein in each fraction were measured by ImageJ and then each signal was divided by the total values for each Ulp2 derivative.
Figure 2(A) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 or MMS. Cell viability was measured after 24 hours using MTT assay. Values are mean ± SD of three assays. (B) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant (E141A) H2AX were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes), subsequently; the cells were cultured in fresh DMEM medium for 2 weeks. The numbers of colony formation were counted. Values are mean ± SD of three assays. (C) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant (E141A). U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes) and harvested at the indicated recovery time points. Then FPG-modified comet assay was performed. NT: no treatment. Representative comet tails were shown. The olive tail moments (OTM) were summarized from at least 50 cells in each experiment. Values are mean ± SD of three assays. (D) DNA replication in U2OS cells expressing the E141A mutant (E141A) is suppressed. Cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes), followed by BrdU incorporation (30 μM in medium, 30 minutes). Representative images show BrdU-positive cells in cells expressing WT H2AX or E141A following genotoxic stress. Nuclei were stained with DAPI (blue). The bottom panel histogram shows the percentage of BrdU incorporation in each sample, Values are mean ± SD of three assays. (E) Generation of AP sites is suppressed in cells expressing E141A. Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes. AP sites were calculated according to the standard curve. Values are mean ± SD of three assays.
Figure 4A, Serum estradiol-17β (E2) levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice (mean ± SEM, Student's t test; n=4 mice for each group). B, Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice. In Rb1d/d mice, pre-implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 (mean ± SEM, Student's t test; n=4 mice for each group). C, Protocol of pre-implantation P4 supplementation for the recovery of epithelial CCA in Rb1d/d mice. Rb1d/d mice underwent subcutaneous injection of P4 (2mg/day/mouse) on days 2 and 3, and sacrificed on day 4. D, Persistent Ki67 expression in the epithelium of Rb1d/d mice on day 4 was recovered by pre-implantation P4 treatment on days 2 and 3. Scale bar = 100μm; le, luminal epithelium; s, stroma. E, Ratio of Ki67-positive epithelial cells in Rb1d/d mice was completely suppressed by pre-implantation P4 supplementation on days 2 and 3. The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2D were used (mean ± SEM, Student's t test; n=5 different mice for each group). Three different high-powered fields per mouse were analyzed and each of the Ki67-posivie ratios was demonstrated. F, Protocol of P4 treatment for the recovery of embryo invasion and subfertility in Rb1d/d mice. P4 (2mg/day/mouse) was injected to Rb1d/d mice on days 2-7 in Group 1, on days 2 and 3 in Group 2, and on days 5-7 in Group 3. G, Resorption rate was reduced on day 8 in Groups 1 (n=41 different implantation sites) and 2 (n=30 different implantation sites), but not in Group 3 (n=40 different implantation sites). The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2I were used. H&E staining was performed using all sections that shows the implantation sites, and resorption rate was evaluated. *P<0.05 vs Rb1f/f mice; **P<0.05 vs Rb1d/d mice; Fisher's exact test. H, Number of pups delivered by Rb1d/d dams was normalized by pre-implantation P4 treatment on days 2 and 3 (Group 2)(mean ± SEM, Student's t test). The same data of Rb1f/f and Rb1d/d dams without P4 treatment in Fig 1E were used. The numbers of pups in 20 Rb1f/f and 37 Rb1d/d dams without P4 treatment and 7 Rb1d/d dams with P4 treatment were evaluated.
Figure 5A-C. 2.5x105 of TGF-β1 siRNA or control siRNA transfected AT-MSCs were intradermally injected around each of CD18-/- murine wound. PBS mock injection served as negative control. Wound tissue were harvested at day 2, 5, and 7 post-wounding for quantification of human TGF-β1 mRNA (A) at day 2 by qPCR, total TGF-β1 (B) and active TGF-β1 (C) protein at day 5 by ELISA. Expression of α-SMA (D, E) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Quantification data are expressed as mean ± SEM, n = 3 wounds per group Expression of CD31 (F, G) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Quantification data are expressed as mean ± SEM, n = 3 wounds per group
Fig.Figure 3A: Ubc9 possesses a specific insertion of two amino acids between the catalytic cysteine and tryptophane W103. Sequences of human UbcH5B (GI: 1145689), human E2-25k (GI: 1381164), human UbcH6 (GI: 1064914), human Ubc12 (GI:4507791), human Ubc9 (GI:4507785), and S. cerevisae Ubc9 (GI:1431070) were aligned using Clustalw with default parameters. The catalytic cysteine is shown in bold. Identical residues are indicated by an asterisk under the aligned sequences. The model of Ubc9 (PDB: 1A3S) was generated using Deepview and rendered using POV-Ray. 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. C: Ubc9 wt and D100A are equally active in CFP-RanGAPtail SUMOylation. Assays were with 35 nM Aos1/Uba2 and 15 nM Ubc9. 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.. 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 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).