EMBO/sd-character-level-ner huggingface dataset:
Siz2 DNA binding activity is correlated with SUMO conjugation to RPA in vitro.Figure 6Fluorescence polarization assays performed with 50 nM 6-FAM-ds20, a 6-FAM-labeled 20 nucleotide double-stranded DNA, and serially diluted Siz2∆CT or Siz2∆CT mutant. Histograms derived from the curves presented in (A) and presenting apparent dissociation constants (Kd) for each Siz2∆CT variant. Histograms derived from the gels presented in (D) and presenting Rfa1 SUMO conjugation rates for RPA complexes in the presence of different Siz2∆CT variants. SUMO conjugation assays performed under multiple turnover conditions as in Fig 4. Experiments were performed in technical triplicates (six times for WT). The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.
Figure 4Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets, followed by western blot analysis of precipitated Parkin and modified Parkin. Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets, followed by Western blot analysis of precipitated Parkin and MsrB2. Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin, LC3 and MsrB2 in HC and DM platelets. Arrows indicate sites of colocalization of Parkin, LC3 and MsrB2 as determined by Volocity software (PerkinElmer, USA). Graphical quantification of colocalization between Parkin, LC3 and MsrB2 signal. Signal intensity of each group was converted to fold and compared with HC values (colocalization of parkin, LC3 and MsrB2; **p=0.0006). Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets. Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies. The detection of MetO Parkin, was performed w/o β-mercaptoethanol (NonRe) SDS sample buffer. In vitro assay. Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs. Western blot analysis of Parkin and MsrB2 was performed. (red arrow, low MsrB2 expression; blue arrow, high MsrB2 expression). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
KZFPs present in the last common ancestor of coelacanth and tetrapods were KAP1 binders A.Figure 6Consensus sequences for KRAB A-Boxes from top to bottom: coelacanth KRAB domains, coelacanth PRDM9 KRAB domain, human PRDM9 KRAB domain and human KRAB domains. Residues conserved between the first three sequences and not present in human KRAB consensus were highlighted in red. In the human KRAB consensus, residues crucial for KAP1 recruitment were highlighted in blue. Immunoprecipitations of HA-tagged KRAB domains in order to check  interaction with cKAP1. Co-transfection of Flag-tagged cKAP1 (upper panel) or Flag-tagged hKAP1 (lower panel) and HA-tagged ZNF398 DUF3669 domain negative control, H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA-immunoprecipitation. cKAP1 and hKAP1 presence was revealed by Western-Blot using an anti-Flag antibody. Input = Cellular lysate, IP = Immunoprecipitate. Western-Blot using an HA antibody on the IPs at the bottom. Evolutionary KRAB model: On top, a simple phylogenetic tree represents the links between echinoderms, coelacanth and human. At the bottom different KRAB domain versions ranging from the ancestral PRDM9-related KRAB domain to the classical KAP1 binding human KRAB are represented. Their position on this model depends i) under which species or clades they were detected, hence reflecting the last common ancestor in which they putatively appeared and ii) whether they bind KAP1, in the blue rectangle, or not, in the pink rectangle. Briefly, the echinoderm-conserved PRDM9 related KRAB domain is the putative ancestor of KAP1-binding KRAB domain that emerged in the coelacanth. The KRAB domain and KAP1 co-evolved, maintaining their association in tetrapods. Meanwhile, certain KZFPs have lost KAP1 binding to become vKZFPs.
Astrocyte diversity in the diencephalon revealed by scRNA-seqA Scheme depicting the improved dissected region (in blue) from 2 - 3 mo old Aldh1l1-eGFP mice (drawing left).Figure 1A Scheme depicting the improved dissected region (in blue) from 2 - 3 mo old Aldh1l1-eGFP mice (drawing left). Schemes to the right depicting the column-based magnetic cell selection using first nano-sized beads for cell depletion of myelin containing material followed by positive cell selection for ACSA-2 cells. Cells isolated by ACSA-2 magnetic associated cell separation were used for scRNA-seq using 10xGenomics strategy. t-SNE projection of 25761 single-cell transcriptomes with clusters color coded and annotated post hoc based on their transcriptional profile identities Cell numbers in the different clusters are: Cluster 0 = 6564, Cluster 1 = 4617, Cluster 2 = 4147, Cluster 3= 4053, Cluster 4 = 1375, Cluster 5 =1033, Cluster 6 = 991, Cluster 7 = 663, Cluster 8 = 633, Cluster 9 = 414, Cluster 10 = 333, Cluster 11 = 222, Cluster 12 = 207, Cluster 13 = 188, Cluster 14 = 154, Cluster 15 = 85, Cluster 16 = 82. For all differentially expressed genes per cluster of scRNA-seq data from diencephalic astrocytes after ACSA2 MACS following an optimized dissection expression of representative astrocyte specific marker genes (numbers reflect scaled log normalized read counts for the specified gene for each cell, levels indicated on the right; purple: high expression, grey: low to no expression). expression of representative ependyma (D) specific marker genes (numbers reflect scaled log normalized read counts for the specified gene for each cell, levels indicated on the right; purple: high expression, grey: low to no expression).
Figure 1A. A schematic showing major SARS-CoV-2 proteins and domain structure of spike protein. B. Changes in the expression of cytokines in THP1 macrophages (left) and PBMCs (right) upon treatment with full-length S1 subunit at 1 µg/ml for 24 hr. Data information, C. Changes in the expression of cytokines in THP1 macrophages upon treatment with different domains of S1 subunit at 1 µg/ml for 24 hr. Data information, data are shown as the mean of three technical replicates; in (D), data are shown as the mean of 20 individual donors. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Multiple t-test. D. Measurement of cytokine release from healthy donor PBMCs treated with PBS or NTD at 1 µg/ml for 24 hr. E. Effect of an anti-RBD antibody on S1 subunit stimulated changes in the expression of cytokines in PBMCs. Left, a schematic showing the domain structure of S1 spike protein and anti-RBD antibody. Right, changes in cytokine gene expression in response to S1 spike protein in the presence or abscene of anti-RBD antibody. Gene expression was measured by qPCR. Cytokine release in the conditioned media was measured by Luminex. Full-length S1 and S1 subunits are purified from HEK293 cells. Data information, data are shown as the mean of three technical replicates; in (D), data are shown as the mean of 20 individual donors. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Multiple t-test.
The lipid requirements for membrane binding by the Atg5–Atg12/Atg16 complex.Figure 2(A) Coomassie‐stained gels of liposome co‐sedimentation assays using either Atg5-Atg12/Atg16 (upper panel) or Atg5/Atg16 (lower panel) and liposomes with the indicated lipid composition (Folch: Folch lipids, PE/PI3P: 40% POPC, 35% POPS, 20% POPE, 5% PI3P, PE/PI: 40% POPC, 35% POPS, 20% POPE, 5% PI, DAG/PI3P: 40% POPC, 35% POPS, 20% DAG, 5% PI3P, DAG/PI: 40% POPC, 35% POPS, 20% DAG, 5% PI). Note that the Atg5-Atg12/Atg16 and the Atg5/Atg16 complexes show almost identical binding behaviours. (B) Quantification showing the amounts of Atg5-Atg12/Atg16 and Atg5/Atg16 in the pellet fractions. The amount of protein in the pellet fraction in the absence of liposomes (background) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes. Data were obtained from three independent experiments, one of which is shown in (A). The Atg5-Atg12 and Atg5 bands were used for quantification. (C) Coomassie‐stained gel of a liposome co‐sedimentation assay showing the lipid requirements of the Atg5-Atg12/Atg16 complex. Liposomes were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. In the liposomes containing no POPE or POPS, these lipids were replaced with POPC. (D) Quantification of Atg5-Atg12/Atg16 in the pellet fraction in liposome co‐sedimentation, one of which is shown in (C). Data were obtained from six independent experiments. The Atg5-Atg12 was used for quantification. The average value is shown and the error bars represent the standard deviation. P‐values were calculated using Student's t‐test. The numbers next to the gels indicate the molecular weight in kDa. DAG, diacylglycerol; P, pellet; PI, phosphatidylinositol; PI3P, phosphatidylinositol‐3‐phosphate; (PO) PC, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylcholine; (PO)PE, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylethanolamine; (PO) PS, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylserine; S, supernatant. Figure source data can be found with the Supplementary data.
Figure.Figure 6A. Experimental scheme for generating dual reporter mice and SCNT B. Representative fluorescence images of siRNA-control or siRNA-6B injected SCNT embryos. Scale bar, 50 μm C. Quantification of embryos that expression tdTomato after injection with siRNA-control or siRNA-6B. Numbers of observed embryos are indicated. n ≥ 3, Error bars, s.e.m., ***P < 0.001 according to two-tailed Student"s t D. Representative images of green fluorescence in Oct4::EGFP reconstructed blastocysts derived from injected siRNA-control or siRNA-6B SCNT embryos. Representative images from ≥ 25 embryos analyzed in three independent micromanipulations are shown. Green fluorescence indicates that the Oct4::EGFP transgene has been expressed. Scale bar, 50 μm E. Oct4::EGFP was up-regulated significantly in ICSI embryos compared with SCNT embryos at blastocyst stages, as determined by RT-qPCR. Data shown are mean expression values relative to Gapdh. The value in ICSI embryos was set as 1. Error bars, s.e.m., n ≥ 3. *P < 0.05, ***P < 0.001 by two-tailed Student"s  t F. Representative image of cloned mice derived by siRNA-6B injected SCNT embryos G. Bar graph showing the efficiency of attachment to the feeder cells (left) and si6B-mdx-ntES derivation (right). The mdx sick mice tail-tip fibroblasts as nuclear donors. N, total number of embryos analyzed for each condition. Results are from four replicate experiments H. Immunostaining images of si6B-mdx-ntES expressed pluripotency markers. Scale bar, 50 μm I. The si6B-mdx-ntES possessed multiple-differentiation potential, as shown in embryoid body.  Scale bar, 100 μm. J. An image of a chimeric mouse derived from si6B-mdx-ntES
Figure 1(A) Phosphorylation of Afadin at Serine1795 (marked with asterisk) revealed by quantitative phosphoproteomic analysis in brown preadipocytes stimulated with IGF-1 for 5 min. Table shows 8 identified phosphopeptides belonging to Afadin, where the phosphorylated residue is underlined and highlighted in bold. (B) Immunoblotting and quantification of brown preadipocytes (WT-1) stimulated with insulin at different doses for 5 min (n=3). The molecular weight corresponded to the I-Afadin isoform. (C) Immunoblotting of mature brown adipocytes (WT-1) stimulated with insulin (100nM) for 5 min (n=4). (D) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min (n=3). (E) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min (n=3). (F) Immunoblotting and quantification of isolated brown adipose tissue (BAT) from male C57BL/6J mice injected with 5U insulin for 5 min (n=4-6). (G) Quantification of immunoblotting of isolated BAT, subcutaneous white adipose tissue (sWAT) and visceral white adipose tissue (vWAT) from male C57BL/6J mice after fasting or re-feeding for 2/6 hrs (n=6). (H) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12-week old C57BL/6 male mice fed with either chow (CD) or high-fat diet (HFD) for 6-weeks injected with vehicle or insulin (n=6). (I) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions (SVF) from lean, obese or Type 2 Diabetic (T2DM) subjects stimulated or not with insulin (n=6). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
Nucleosome patterning associated with the RE1 NRSF/REST binding motif.Figure 4A The average distribution of nucleosomes centred on the RE1 site (n=871), spanning 2 Kb of flanking genomic sequence. B, C Aligned pair-end sequence read data were stratified into two size classes, (B) 122-137 bp and (C) 162-188 bp for pl-iPSC and NPC. Plots show the average distribution of nucleosomes centred at and surrounding the RE1 site (n=871). Peaks correspond to the NRSF/REST complex, and nucleosomes in B and C respectively. D Cluster analysis of nucleosome positioning centred on the RE1 site ± 300 bp. RE1 sites were clustered based on the 122-137 bp mid-point sequence read values (NRSF/REST protein complex) from pl-iPSC, shown as a frequency distribution in upper left panel. The frequency distribution data for the same sites was plotted using 122-137 bp data from NPC (upper right panel) and 162-188 bp data (nucleosomes; bottom panels) for the strongest (solid line) and weakest (dotted line) clusters. E Schematic of nucleosome patterning at the RE1 site in pl-iPSC and NPC. Nucleosomes are represented by filled black circles.
Ketone bodies shift T cell metabolism to oxidative phosphorylation.Figure 2Human PBMCs were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan T cells, CD4+ and CD8+ T cells were isolated through magnetic cell separation. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. (A-C) OCR, maximum respiration, spare respiratory capacity and basal respiration were measured in A pan T cells, B CD4+ T cells and C CD8+ T cells, n=9 (pan T), n=3/4 (CD4 unstimulated), n=7/8/9 (CD4 stimulated), n=5 (CD8 unstimulated) and n=8/7/7 (CD8 stimulated) individual experiments. Data depicted as mean ± SEM (OCR) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
Figure 3A-B MAVS could be succinylated in vitro. GST-MAVS (A) extracted from E.coli or Flag-tagged MAVS (B) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. Protein succinylation was detected with anti-pan-succinyl-lysine antibody; GST-MAVS or Flag-MAVS was stained by Coomassie blue. C The succinylated residue in MAVS was identified by mass spectrometry analysis. D Sequence alignment of partial MAVS (1-30 amino acids) from human, macaque, cow, pig, dog, mouse and rat. The red box indicates a conserved lysine (K7). E Dot-blot assay for the specificity of anti-succ-K7-MAVS antibody. Equal amounts of succinyl-peptides or the control peptides were immunoblotted with the indicated dilutions of anti-succ-K7-MAVS antibody. GST-MAVS (F) extracted from E.coli cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody. Flag-MAVS (G) purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody. H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5-intact H1299 cells (SIRT5 +/+) (3.2 vs. 1.0). The cell lysates from SIRT5-deficient H1299 cells or the SIRT5-intact H1299 cells were immunoprecipitated with anti-MAVS antibody or mouse IgG control, followed by immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates. I Reconstitution of WT SIRT5 in SIRT5-deficient H1299 cells (SIRT5 -/-) caused a significant reduction in succinylation of MAVS at Lys 7 (1.0 vs. 0.2); but overexpression of SIRT5-H158Y in SIRT5 -/- H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 (1.0 vs. 1.2). The SIRT5 -/- H1299 cells were transfected with Flag-SIRT5 or Flag-SIRT5-H158Y, followed by immunoprecipitating with anti-MAVS antibody or mouse IgG control, and immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates. J-K Knockout of Sirt5 increased Mavs succinylation in mouse livers (J) and lungs (K). Proteins extracted from livers (J) and lungs (K) of Sirt5 KO and the wildtype littermates (n = 3 per group) were detected by the indicated antibodies. MAVS succinylation was determined by anti-succ-K7-MAVS antibody, and anti-pan-succinyl-lysine antibody was used as positive controls.
DNA duplex processing by Sgs1 A Sketch of the employed magnetic tweezers assay and DNA processing by Sgs1.Figure 1A Sketch of the employed magnetic tweezers assay and DNA processing by Sgs1. B Observed dsDNA processing patterns of Sgs1, including an overview over consecutive bursts and pauses (upper panel) as well as detailed views into individual bursts containing multiple unwinding events (lower panels). A typical unwinding event of Sgs1 starts with slow, gradual unwinding of the dsDNA followed by DNA rehybridization, that can be almost instant (76% of events, see blue sections in lower right panel) or contain slow rewinding sections (24% of events, see orange sections in lower right panel). C Histograms of the observed unwinding and rewinding velocities for Sgs1. The mean unwinding rate was 65 ± 2 bp/s (N = 899). The mean rewinding rate was 115 ± 6 bp/s (N = 287). We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections.
Ribosomes stalled on poly(A) stretches reveal alterations in both the peptidyl-transferase and decoding centers.Figure 5(A, B) Schematic representation (A) of the stalling situation on poly(A) tract mRNA. Cryo-EM density map of the poly(A) stalled 80S RNC filtered according to local resolution and used to build the molecular model (B). (C) Cryo-EM density (mesh) and stick model with cartoon phosphate backbone representing the peptidyl-tRNA in the peptidyl-transferase center (PTC). (D) General overview of the A, P, and E sites with the P/P tRNA and mRNA. (E, F) Detailed view of the mRNA in the A site using sticks model with cartoon phosphate backbone representation and cryo-EM density (mesh). The poly adenine mRNA sequence forms a π-stacking array between positions +4 and +7, which is stabilized from both sides by stacking of 18S rRNA bases C1634 and A1756.
Figure 1 Figure 1. DENND2B binds ITSN  Purified GST-SH3-containing proteins were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.  Purified GST-SH3 domains of ITSN were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. Lysates from HEK-293T cells expressing Flag-ITSN-s and GFP or GFP-DENND2B were incubated with protein A beads or protein A beads coupled to anti-GFP. Bound proteins were detected by Western blot. D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively. D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.
Figure 7Corrosion casting results showing the linearization of capillaries in IGF2R-treated VM mdx muscles. Pericytes around capillaries are shown in the insert box (a). Scale bars=100μm. Representative immunofluorescence results (b) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown. Representative immunofluorescence quantification (c) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown. Scale bars=75μm. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean± SEM. (d) Representative WB showing CD146 and NG2 expression in total protein lysates of TA and VM muscles of mdx mice treated with anti-IGF2R. Densitometry analysis of data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean ± SEM. (e) Representative Ve-cadherin (red) and CD31 (green) expression in small vessels of VM muscles from untreated mdx and mdx treated with low dose of anti-IGF2R for 4 weeks. Scale bars=25 μm.
Figure 6A Control animals (+/w; UAS-sec5IR/+), n= 19, and those with salivary gland-specific knockdown of sec5 (fkh-GAL4/w; UAS-sec5IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-sec15IR/+), n= 20, and those with salivary gland-specific expression sec15 (fkh-GAL4/w; UAS-sec15IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (+/w; UAS-sec3IR/+), n= 20, and those with salivary gland-specific expression sec3 (fkh-GAL4/w; UAS-sec3IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.G Control animals (+/w; UAS-sec8IR/+), n= 20, and those with salivary gland-specific knockdown of sec8 (fkh-GAL4/w; UAS-sec8IR/+), n=19, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.I Control animals (+/w; UAS-exo84IR/+), n= 19, and those with salivary gland-specific knockdown of exo84 (fkh-GAL4/w; UAS-exo84IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.J Quantification of data from (I). Data are represented as means. Statistical significance was determined using a Chi-square test.