EMBO/sd-character-level-ner huggingface dataset:
B HeLa cells were treated with control (CTRL), SEPT2, SEPT7, SEPT9, Drp1, or SEPT7+ Drp1 siRNA for 72 h, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Graph represents the distribution in length (μm; whiskers from min to max) of mitochondria in siRNA-treated cells from 3 independent experiments (analysis of at least 100 measurements per biological replicate). Student's t-test, *** = p<0.001.
F Representative protein expression of EMT inducers in pancreatic lysates prepared from KNCmice with different tumor gradings.
(A) Hematoxylin and Eosin (H&E) staining of WT and K14ΔNLef1 HF from back skin at 11.5 weeks (early anagen). White arrowheads: ectopic SG and epidermal cysts. Data information Scale bars: 50 μm.
G. The hierarchy of Yb body assembly proposed in this study. Domain structures of Yb, Armi, SoYb, and Vret are shown on the right. Hel-C, Helicase C-terminal; eTud, extended Tudor; ZnF, zinc finger; RRM, RNA-recognition motif; MYND, MYND-type zinc finger.
FRAP of Tau accumulations show no recovery of NUC Tau (NUC: <10% recovery; t1/2 ≈11 s), ~20% recovery of CYT and NE Tau with a faster recovery rate for NE Tau (NE: t1/2 ≈2.5 s; CYT: t1/2 ≈7 s). N=15-20 Tau accumulations per type. Data shown as mean±SD.
Representative of total cell lysate and the corresponding densitometric analysis of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.
(A) RNA expression profiles of first line ROS defense systems in V. cholerae monitored by S1 nuclease mapping assay. Quantified average values (normalized to WT before PenG exposure) from three independent experiments are presented under the signals of each gene. White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material. Note that the loading control is the same as in Fig. 2D, as these S1 assays were done on the same RNA preparations.
(G) Time from Start to degradation of 50% of the indicated Far1-GFP sensor in single cells. The bars show median ±95% confidence intervals. The numbers above the plot show median values. **** indicates p-value <0.0001, *** <0.001 and ns >0.05 for pair-wise comparisons with wild-type using Mann Whitney U test.
Colony size of SCC13 Act cells (clone 2) after co-culture with either immortalized mDia2 knockdown (sh-mDia2) or control (sh-EV) mouse fibroblasts. N=4.
(A) Experimental set-up for administrating control liposomes (Ctrl-Lipo) or macrophage-depleting clodronate liposomes (Clod-Lipo) as well as vehicle control (Veh) or Tamoxifen (Tam) for inducing whole body NLRP3A350V expression in Nlrp3iR26-A350V/WT mice.
(e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a.
Representative nuclease assays as in (A), but with a substrate containing a nick in the top oligonucleotide (20 nt away from the 3'-end, see cartoon).
(B) Confocal images of RGC inputs traced by CTB488 and mCherry. CTB488+ RGC inputs in the Cdc50a-RNAi area overlapped with vGluT2. Scale bar, 20 µm.
A, B, Joint analysis of nonCM scRNA-seq data from uninjured hearts and hearts at 2, 7, and 14 dpi with LIGER. A, All nonCM visualized on tSNE. B, top, NonCM from each time point visualized on the tSNE embedding in (A). Data was down-sampled to the same cell number at each time point. B, bottom, Pie charts showing contribution of different cell types at each time point. C-F, Zoom-in analysis of macrophages identified in (A).
cell cycling status of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice.
(C) Fluorescence polarization assays demonstrating that a peptide containing the BH3‐like domain of Beclin‐1 interacts with Bcl‐XL. Recombinant Bcl‐XL ΔTM was incubated with carboxyfluorescein‐labeled Bak BH3 peptide, in the absence or presence of wild‐type (WT) Beclin‐1 BH3 or either of the two mutant peptides described in B.
Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (B) Quantification of CFU of C. albicans per gram tongue tissue of the indicated mice at day 7 p.i. Data are mean ± SEM from 4 repeat experiments and 12 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.
(A) IPA analysis of RNA-seq data showing the 14 top-predicted upstream regulators of differentially expressed genes. Positive Z-scores (orange) represent activated upstream regulators, negative Z-scores (blue) represent inhibited upstream regulators.
(E) Epifluorescence image of an M45-expressing cell with large GFP-LC3 structures
I. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in control cerebellar organotypic slices (b-d), and in cerebellar slices at 10 dpl in absence (f-h) or in presence of 100 μM D-Asp (j-l). Panels d, h, and l display colocalized points (white). Panels a, e, and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a, e ,i: 200 μm; in b-d, f-h,and j-l: 50 μm
(G) Ezrin protein expression in ezrin knockdown HBMEC using shRNA (left panel) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC (right panel).
J. Quantification of the impact of rIFN-β on Drp1 phosphorylation after silencing of genes encoding the kinases CDK5, Camk2a, and ERK2 and the phosphorylase PGAM5.
Quantification of HW/TL ratio (H), N=8 to 13 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice. *
Volcano plot representing the mass spectrometry signals from 71 glycerophospholipids, namely 49 PC (phosphatidylcholine), (39 non-plasmalogens and 10 plasmalogens), 14 PE (phosphatidylethanolamine), 5 PS (phosphatidylserine), and 3 PA (phosphatidic acid), which are shown as different color symbols as indicated, analyzed in gradient-purified mitochondrial extracts from subject vs. control cells. All lipid entities shown above the horizontal red line have a p-value < 0.05.
(B) Cells sense contact with proximal cells through a cascade dependent on the presence of TslA and OmpA. Possible interaction of TslA with TssM triggers membrane complex/baseplate assembly followed by the formation of a few sheath rings. TssA caps such sheath foci for further polymerization upon unknown signal. The Hcp tube and VgrG spike along with effectors are propelled out of the cell during sheath contraction. The ATPase ClpV then disassembles the contracted sheath.
(B-D') A temporal expression profile of TH at 48, 72 and 96h AEL lymph glands in control background (domeMESO-Gal4, UAS-GFP/+) (E-G') A temporal expression profile of Ddc at 48, 72 and 96h AEL lymph glands in control background (domeMESO-Gal4, UAS-GFP/+). (H) Quantification of TH levels (domeMESO-Gal4, UAS-GFP/+, n=18 at 48h, n= 15, at 72h and n= 23 at 96h AEL). (I) Quantification of Ddc levels (domeMESO-Gal4, UAS-GFP/+, n= 17 at 48h, n= 33 at 72h and n= 38 at 96h AEL). Data information: DNA is stained with Hoechst in blue, iDopamine in red, Tyrosine hydroxylase (TH) and Dopa decarboxylase (Ddc) in red, domeMESO (representative of progenitors) expression in green, AEL indicates After Egg Laying. The quantifications in H, I represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in H, I, is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.
Structural modelling of ATG16L1 residues 160-205 (magenta helix) in the presence of lipid bilayer (green lines). Highlighted residues include I171, K179 and R193 as sticks, which are mutated in this study. Shown in yellow stick, a PI3P molecule embedded in the lipid bilayer and interacting with some of the positively charged residues of ATG16L1.
F) Proportion of TUs per TSS type. a-gTSS are by definition excluded from the analysis since they are facing the nearest downstream gene.
(B) ACE2 protein abundance in different human tissues from ProteomicsDB, with the median expression presented in log10(iBAQ).
, Simulated inactive enzyme. The inactive enzyme is the sum of the enzymes that are synthesized but do not carry fluxes. The production of the inactive enzyme indicates that total proteome is not constrained. The grey area represents where the proteome switch occurs in experiments (Goel et al, 2015), which is between 0.5 and 0.6 h-1.
(A) Validation of WASH and retromer knockouts and SLC3A2 protein levels. Immunoblots of parental or FAM21, VARP and VPS29 knockout cell populations.
TNF-dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype measured by DEVD-AFC. MEFs treated with hTNF (100ng/ml) alone, or with CHX (1µg/ml) for 4 hours (H) or 2 hours (I) subjected to the Caspase 3 activity assays. A representative data of two independent experiments, n=4.
CRISPR-based detection method for novel coronavirus. General scheme of CRISPR detection procedure. In the two strategies all the process takes less than 60 min.
Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Blood glucose levels and AUC during glucose tolerance tests. Blood glucose levels and AUC during insulin tolerance tests. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP). Percentage of suppression of hepatic glucose production (HGP).
A. Expression instability of the chicken BU-1 locus as a reporter for replication impediments formed by structure-forming DNA sequences. The leading strand of a replication fork entering the locus from the 3" end encounters a DNA sequence with structure-forming potential located 3.5 kb downstream of the transcription start site. In wild type cells this is a G4 motif, which is replaced by (GAA) n repeats in this study. Under conditions in which polymerase stalling is prolonged, e.g. loss of G4 processing enzymes or G4 stabili or repriming is defectiv the persistence of a putative ssDNA gap leads to a zone of interrupted histone recycling and loss of parental histone modifications. If this loss of modifications involves a control region of the gene, e.g. the promoter, it can result in a change in expression
The effect of TRIM21 on hSAMHD1 mutants. HEK293T cells were transfected with VR1012 or TRIM21 and SAMHD1 mutants for 48 h and then subjected to IB analysis. Tubulin served as a loading control.
D Each HA-tagged PB1-F2 was transfected into 293T cells with or without Flag-ubiquitin. At 18 h after transfection, cells were treated with MG132 for 6 h. The lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting with anti-Flag antibody. PR8, A/Puerto Rico/8/34 (H1N1); 1918, A/Brevig Mission/1/1918 (H1N1).
(A, B) Whole-mount immunostaining and quantification for p53 on Ctrl and Sas-4−/− embryos at E7.5 (A) and E6.5 (B). Representative sagittal planes are shown and the dotted lines demarcate the epiblast. Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below, normalized to control embryos in the same batch at E7.5 (4 embryos) and at E6.5 (9 embryos). *** p < 0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bars = 100 μm. (A) Ctrl: 1.04 ± 0.00 (n=495 cells); Sas-4-/-: 1.65 ± 0.19 (n=850). (B) Ctrl: 1.00 ± 0.18 (n=1665); Sas-4-/-: 1.16 ± 0.44 (n=1330)
(I) Control and CEP55 knockdown MDA-MB-231 cells were synchronized as above, released into culture medium for six hours and then blocked with nocodazole (0.5 µM) for an additional four hours prior to treatment with the Cdk1 inhibitor RO-3306 for an additional 16 hours. Sub-G1 cells were then identified by propidium iodide staining and quantified by FACS. Graph represents the mean±SEM of two independent experiments.
(C) Gross appearance of the full-length mouse colon. Each line on a ruler represents a millimeter (mm).
The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP, E-cadherin, and β-tubulin antibodies (G). C: cytoplasm. M: cell plasma membrane.
(C-D) Phosphopeptide abundances (log2 ratio WT [HU/untreated]). Colored dots are factors with significantly different phosphopeptide scores WT [HU/untreated] <4 (blue) or >4 (red) and p value<0.05 (D) Amongst the phosphopeptides induced on HU, factors involved in transcription are highlighted (green dots, n=87).
measuring fibroblast velocity (C), directionality (a cell with a directionality equal to 1 is moving straightforward) (D) and migration distance (E) or tumour cell velocity (F), directionality (G) and distance (H). Values are means ± SEM of 16 tracked cells from two independent experiments.
b Grin2aS/S mice showed the clasping reflex. c During the 12 h dark phase on the Lafayette running wheel the running distance is reduced in Grin2aS/S mice.
(b) Summary of the mapped locations of integrations in the genomes of two F1 hybrid mES cell clonal cell lines. LAD and iLAD integrations are shown in orange and black, respectively.
MDM were treated with 5μM ETO for 18h and infected with HIV-1 BaL (3, 6, 12 ffu/cell). Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ** P‐value ≤ 0.01; ***P‐value ≤ 0.001, paired t‐test).
H Exogenous supply of membrane-permeant PI(3) P acutely rescues defective Synaptophysin-pHluorin endocytosis induced by pharmacological inhibition of VPS34-mediated PI(3) P synthesis in hippocampal neurons. Data are similar to those shown in C and D; where indicated exogenous PI(3) P-AM (20 µM) was supplied in parallel to VPS34IN1 (10 µM) for 1 h prior to imaging. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
C Analysis of mitochondrial morphology in electron micrographs (left image: scale bar, 1 μm; right image: scale bar, 0.1 μm).
K Western blot analysis of ubiquitination of cellular proteins (Pan-Ubi) in p97-WT or p97-KO HEK293T cells treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.
(A, B) Flag‐SENP3 or HA‐PELP1 was expressed in HeLa cells as indicated. Proteins were captured on Flag‐ or HA‐beads and immunoblotted using antibodies directed against endogenous PELP1 or SENP3 as indicated.
Coomassie- stained proteins from bovine retina that were co-immunoprecipitated by the anti-RIBEYE antibody 2D9 (lane 3). Lane 2, control precipitation by irrelevant mouse IgGs; lane 1, input (10% of the total loaded in each lane) Western blot of the immunoprecipitated proteins (as in A, 1% of the total protein was loaded per lane) probed with anti-RIBEYE (Ba), anti-CASPR1 (Bb) and anti-CNTN1 (Bc). The 2D9 antibody immunoprecipitated RIBEYE (Ba, lane 3), CASPR1 (Bb, lane 3) and CNTN1 (Bc, lane 3); these proteins were not detectable in the control immunoprecipitations. Bd) As a loading control, heavy chains of the precipitating antibody (IgG hc) were visualized by the goat anti-mouse IgG antibody conjugated to peroxidase
iBMDMs stably expressing GFP-PLK4 and parental iBMDMs were unstimulated or stimulated with 0.5 ug/ml LPS for 4h. The PLK4 interaction with endogenous CYLD was analyzed by co-immunoprecipitation with anti-GFP antibody.
Immunofluorescence analysis of Myc-Egg K987A and Myc-Egg R988A mutants (red) in OSCs. Both mutants were fused to SV40-NLS. Anti-Myc antibody was used for detection. DAPI (blue) shows the nuclei. Scale bar: 10 μm.
(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. RA-target gene expression was assessed at day 12 (section v).
IFN-α induced expression of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. Protein expression was determined Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.
I-K The properties of miR-1236-/+ and miR-1236-/- KGN cells versus control KGN cells were assessed by measuring cell viability (I), cell proliferation (J), and cell migration (K). The data are presented as the mean ± SEM of three independent experiments. The migrated cells were imaged under a bright-field microscope (100 × magnification; top of Fig. 6K). Scale bar = 100 µm.
Metagene profile of plaNET-seq mean signal over whole genes (length from 0.5 Kb to 5 Kb, scaled to 500 bins, n=27042) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).
Representative immunofluorescence images of gastric units after vehicle or 72h high-dose tamoxifen (HDT) treatment stained for GIF (red, granules), GSII (green, neck cells and SPEM marker), BRDU (white, proliferation) and DAPI (blue, nuclei). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowheads point to areas of base dropout where ZCs have been lost. Scale bar, 50μm. Stomach units outlined by dashed white line.
(g) Oscillations in the total citrate concentration throughout the cell cycle (mean and s.d. of n=5).
A. Angptl2 mRNA levels in small intestine (proximal, mid and distal), cecum and colon (proximal and distal) of wild-type mice based on qRT-PCR analysis. n=5.
(A) HeLa cells were transfected with the indicated siRNAs. Seventy h after transfection, the lysates from the cells were subjected to immunoblot analysis using anti-TDP-43, DHC, DIC, Dynactin 1 and GAPDH antibodies. The relative densities are shown on the right side. Data from three independent experiments were represented as means ± S.E.M., ns, not significantly different; **, p<0.01, one-way ANOVA.
A-D Quantification of the average expression ± s.e.m. of Dpcr1 and Cd55b mRNAs (A, C, E) or Fabp1 and Ace2 mRNAs (B, D, F) in jejunal spheroids cultured in differentiation medium containing EP4i or dmPGE2. (A, B) Gene expression analyzed 2, 6, 12 or 24 hours after the start of treatment (n = 3 independent experiments). ***p<0.001, ****p<0.0001 comparing the two treatment groups and †p<0.05, †††p<0.001, ††††p<0.0001 compared to the 2 hr time point of the same medium by 2-way ANOVA and Sidak's multiple comparisons test. (C, D) Gene expression was analyzed after culturing spheroids in EP4i (E) or dmPGE2 (P) for the first 12 hours followed by wash out and re-feeding with EP4i or dmPGE2 for the second 12 hours as shown (n = 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by one-way ANOVA and Dunnett's post-test.
Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets undergo fusion events upon light induction. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets.
(D) schematic representation of circSLC8A1 with Ago2 binding sites from CLIP-seq and predicted miRNA binding sites.
Changes in NL interactions after CRISPRa activation of MLK4 in human RPE-1 cells. Visualization of DamID data as in Figure 2a-b.
(C) Complexome analysis of MEFmitochondria (n=3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN-PAGE analysis are shown after hierarchical clustering.
(g) Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 receptor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin.
D) Heatmaps depicting top 25 enriched genes based on adjusted pvalue and log2 fold changes in small airway cells after SARS-CoV-2 infection. Colored bars represent Z-scores of log2 transformed values.
A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for bfr-Esr41 (H). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.
CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age and recordings were made at least 4 weeks after viral delivery on neurons with NachBac (one side) or control neurons (contralateral side) of the same animal (A);
H. Rabl2Q80L interacted with the IFT74/81 heterodimer. FLAG-tagged Ift74 and Ift81 and GST-tagged Rabl2Q80L and Rabl2S35N expressed in E. coli were mixed as indicated and pulled down with glutathione-conjugated beads. Antibodies to GST and FLAG were used for immunoblotting.
In vitro growth of crypts (small intestine) from mice as indicated at day 5 post-induction without R-spo1. Quantification of sphere-forming efficiency of AhCreERCatnblox(ex3)/+AhCreERCatnblox(ex3)/+Cdh1fl/+, day 5 post-induction. N = 3 mice per genotype, mean of 6 technical replicates per mouse, P = 0.04 one-sided Mann-Whitney U-test.
(D) Vertebrate TLSs contain fewer uORFs than expected. Histograms show the distribution of Z-scores in zebrafish, mouse, and human TLSs, with positive z-scores indicating uORF enrichment and negative z-scores indicating uORF depletion, relative to sequence-shuffled TLSs.
B. 293T cells were transfected with control or USP9X-targeting siRNAs. One day after siRNA transfection, the cells were co-transfected with CMV-renilla, 8X-TBS luciferase, Flag-YAP and HA-TEAD2. Reporter activity was quantified as described in (A) (n=4). Error bars indicate the S.E.M. (***p < 0.001; paired Student's t-test).
(A) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation-induced autophagy. HeLa cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT). 72 hours later, cells were incubated with XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented (or not) with inhibitors for 2 hours. Cells were fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation
Immunoblots analysis of CEP55 protein expression in MCF10A cells stably transfected with a Flag-CEP55 expression construct and two isogenic lines were obtained (CEP55-#16 and CEP55-#17) with its respective empty vector (EV) transfected parental cells. COX-IV served as a loading control.
(A) Time-course SUMO conjugation reaction in the presence or absence of ssDNA (virion ϕx174) using recombinant full-length Smc5-Nse2 complex. The substrate utilized was the C-terminal kleisin domain of Nse4 (cNse4). Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer. Labels indicate the bands in the SDS-PAGE of the proteins in the reaction mixture (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2 and N-3S2, cNse4-3SUMO2)