EMBO/sd-character-level-ner huggingface dataset:
Figure 4A. (left panel) Cdk-dependent in vitro kinase assay of Apc1-loop300. 3xFlag-tagged WT or 7T Apc1-loop300 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 30˚C for 30 min, and separated by SDS-PAGE and detected by autoradiography. (right panel) 32P-phosphorylated WT or 7T Apc1-loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS-PAGE followed by autoradiography. B. Quantification of removal of radioactivity in A. Error bars, s.e.m. from three independent experiments. C. Cdc20-5A efficiently binds to the B56 binding site mutant APC/C. The purified recombinant WT APC/C or Apc1-loop500 mutant APC/C (1-L557A/V560A) was incubated with WT Cdc20 or non-phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23˚C for 55 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. D. Quantification of C. The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments. E. The APC/C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A-B56γ at 23˚C. Samples were taken at the indicated time points and analysed by SDS-PAGE and immunoblotting with antibodies, including phospho-site specific antibodies for pCdc20 (pT79) and pApc1 (pS314/pS318). F. A model for Apc1-loop500-mediated Cdc20-APC/C complex formation in mitosis. Apc1-loop500 is phosphorylated by Cdk1 and binds to PP2A-B56 in anaphase. PP2A-B56 dephosphorylates inhibitory phosphorylation sites in N-Cdc20 and promotes the formation of active APC/C-Cdc20 complex. Apc1-loop500 may control phosphorylation of other APC/C subunits and possibly interacting proteins although the mechanisms involved remain elusive. The indicated numbers on the schematic view of the APC/C (the back view is rotated by 180˚ around the vertical axis from the front view) represent APC/C subunits.
Figure 7(A) HEK293T cells co-transfected with YFP-p38IP and Flag-vector, Flag-USP3 or Flag-USP4 as indicated were lysed and immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies. (B) HEK293T cells transfected with Flag-USP4 and YFP-vector or YFP-p38IP as indicated were lysed and immunoprecipitated with anti-IgG or anti-TAK1 and then immunoblotted with anti-Flag and anti-TAK1. (C) HEK293T cells transfected with plasmids as indicated were lysed and immunoprecipitated with anti-TAK1, followed by in vivo ubiquitination assay of TAK1 as described in methods. (D) In vitro deubiquitination assay of TAK1 as described in methods. (E) sh-NC and sh-p38IP Jurkat E6.1 cells transfected with si-NC, si-p38IP, Flag or Flag-USP4 as indicated were stimulated with anti-CD3/CD28 for 0 and 15 min at 48 hours post transfection, and then lysed and immunoprecipitated with anti-TAK1 under stringent conditions and immunoblotted with anti-ub. Statistical analysis of p-p38 levels was shown below the blot. **P < 0.01; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.). (F) Jurkat E6.1 T cells were stimulated with anti-CD3/CD28 for the indicated times. The cell lysates were immunoprecipitated with anti-p38IP or anti-TAK1 and then immunoblotted with the indicated antibodies. (G) sh-NC and sh-p38IP Jurkat E6.1 cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and then lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-USP4 and anti-TAK1. (H) Mapping the USP4 binding domain of p38IP. (I) Overall structure of p38IP U4B (gold) and USP4 (deep teal). (J) Structural comparison between CYLD (blue) and USP4 (deep teal), the fingers subdomain was shown in red. (K) Schematic illustration of the molecular mechanism underlying p38IP regulation of TAK1. In quiescence state, p38IP constitutively competes with TABs for binding to TAK1. Upon receptor stimulation, p38IP transiently dissociates from TAK1, and increases its association with USP4, which might due to the phosphorylation of p38IP (indicated by an asterisk in the figure). Once TAK1 is activated, p38IP not only reassociates with TAK1, disassembling TABs-TAK1 complex and blocking the transfer of unanchored K63-linked polyUb chains from TAB2 to TAK1, but also specifically recruits USP4 to remove the unanchored K63-linked polyUb binding or conjugated ubiquitination of TAK1.
a) MN-1 cells were transfected with mCherry-EGFP-LC3 vector, fixed and imaged after 24 h.figf1(a) MN-1 cells were transfected with mCherry-EGFP-LC3 vector, fixed and imaged after 24 h. Numbers of yellow puncta (autophagosomes) and red puncta (autolysosomes) per cell were counted. Autophagosomes: n = 3 independent experiments, F = 3.56; autolysosomes: n = 3 independent experiments, F = 20.13. One-way ANOVA with post hoc Tukey test. *P 0.05, ***P 0.001. n = 34 cells per genotype. (b) We transfected MEFs stably expressing mCherry-EGFP-LC3 with BFP-tagged AR25Q or AR125Q. Cells were imaged after 24 h, and yellow puncta (autophagosomes) and red puncta (autolysosomes) in AR-expressing (blue) cells were counted. Autophagosomes: n = 3 independent experiments, F = 20.65; autolysosomes: n = 3 independent experiments, F = 4.49. One-way ANOVA with post hoc Tukey test. *P 0.05, **P 0.01, ***P 0.001. n = 33 cells per genotype. (c) MN-1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux. A representative LC3 western blot is shown. All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (d) The ratio of LC3-II:actin for c determined by densitometry analysis using ImageJ. n = 3 independent experiments, F = 0.4501, one-way ANOVA with post hoc Tukey test. *P 0.05. (e) MN-1 cells were treated with ammonium chloride to block lysosomal activity, and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6-h recovery period. We performed p62 western blot analysis on MN-1 cells at baseline (-), after ammonium chloride treatment (+) and after the recovery period (Rec). All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (f) On the basis of the p62 values obtained in e, we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline/recovery values to baseline/recovery for MN-1 WT cells. n = 3 independent experiments, F = 5.45, one-way ANOVA with post hoc Tukey test. *P 0.05, ** P 0.01. Data are presented as mean ± s.e.m. Scale bar, 20 μm. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.
Fig.Figure 4A. MDCK cells were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated. B. The intensity of the bands was measured in three independent experiments. C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. D. Quantification of the NTA data obtained in five independent experiments. E. Working model of polarized sEV release from MDCK cells. Apical release of sEVs (mainly CD63-enriched, CD9-positive, Annexin I-negative exosomes) and basolateral release of sEVs (mainly CD9-enriched, CD63-positive, Annexin I-negative exosomes) are mediated by ALIX-Syntenin1-Syndecan1 and ceramide, respectively. Marker sizes on sEVs (colored circles) reflect the abundance of each exosome protein. Ceramide also contributes to apical release of uncharacterized "CD9/CD63-negative sEVs".
Steady state levels of p62 decrease during poliovirus infection.Figure 1(A) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at the indicated hours post-infection (h.p.i.). Three independent experiments were measured for quantification of p62 levels, and the blot shown is representative. (B) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at 6 h.p.i. Cycloheximide (Cy.) was added to mock infected cells at a final concentration of 50 µg/mL, for 4 hours. Cells were then pre-treated with 0.1 µM bafilomycin A1 (Baf.A1) for 14 hours prior to infection and kept under treatment throughout infection. MG132 was used at a final concentration of 20 µM and added to the media at the time of infection. The blot shown is representative of three independent experiments.
Ccno is specifically expressed in multiciliated cells and the embryonic node A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization.Figure 1A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization. Occasionally, additional expression is observed at the posterior tip of the embryo (asterisk). Double labelling by staining for LacZ expression from the CcnoTA allele (shown in B) and in situ hybridization for Dand5, marking crown cells at the circumference of the node. Ccno expression is restricted to ciliated, ventral pit cells of the node as detailed in the transverse section at the indicated plane. B Schematic of the CcnoTA targeted LacZ knock‐in allele used in (A) and (C-G). C Whole embryo X‐Gal staining of E16 CcnoTA/+ and wild‐type control embryos and indicated section planes of (D-F'). D-F' Histological sections of LacZ‐stained CcnoTA/+ embryo at E16 reveal Ccno expression in the epithelium of (D) plexus choroideus and ependyme, (E) snout epithelium, and (F, F') trachea and bronchi. pc, plexus choroideus; se, snout epithelium; tr, trachea; and br, bronchus. G Co‐expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X‐Gal and immunohistochemistry using a FOXJ1‐specific antibody.
a) Immunoblot analysis of lysates of human monocytes-macrophages primed with LPS (10 ng) and then transfected with poly(dA:dT) (1.figf7(a) Immunoblot analysis of lysates of human monocytes-macrophages primed with LPS (10 ng) and then transfected with poly(dA:dT) (1.5 μg/ml) or treated with nigericin (4 μM) for 6 h (numbers below lanes as in Fig. 1d). (b) Immunoblot analysis of processed caspase-1 and IL-1β (top) and enzyme-linked immunosorbent assay of IL-1β (middle) in supernatants of monocytes-macrophages treated for 6 h with various combinations (bottom) of poly(dA:dT) (b) or nigericin (c), plus 3-MA and amino-acid starvation. *P 0.01 (unpaired t test). (d) Confocal microscopy of LPS-primed human monocytes-macrophages transfected for 3 h with poly(dA:dT) (top), treated for 3 h with nigericin (middle) or left unstimulated (bottom), immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins. Second row, three-dimensional volume rendering reconstructed from confocal z-stack images. Scale bars, 10 μm. Data are representative of at least two experiments.
Figure 3(A) LN229 cells stably transduced with Flag-tagged Merlin or empty vector were treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by mass spectrometry. Numbers of unique peptides of each protein in the immunoprecipitated products are shown. (B) LN229 cells transduced with a pool of four siRNAs against NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot. (C) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. (D) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization (see Methods) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. (E) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA. These cells were detached as in (D), lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot. (F) HEK293T cells were transfected with indicated genes and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. (G) HEK293T cells were transfected with indicated genes. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot. (H) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody, a NEDD4L antibody, or IgG. The immunoprecipitated products were subjected to western blotting.
Figure 2Experimental design for isolating Scgb1a1 lineage-labeled cells from control and Lats1/2 dKO lungs at indicated time points post tamoxifen treatment. Isolated cells were subjected to scRNA-seq analysis. Clusters of Scgb1a1 lineage-labeled cells from 10XGenomics 3' single-cell RNA-sequencing (scRNA-seq) analysis visualized by Uniform Manifold Approximation and Projection (UMAP), assigned by specific colors at day 5 and 14 post tamoxifen treatment. The number of cells in the individual cluster is depicted in the figure. Distribution of each cluster across indicated time points after tamoxifen treatment. Bar graph showing the percentage of secretory, DATP, and AT1 cell clusters in control and Lats1/2 dKO lungs at day 5 and 14 post tamoxifen treatment. Blob plot depicting selected marker genes in cell clusters. Dot size encodes the percentage of cells expressing the gene, and color encodes the average per cell gene expression level. Pseudotime ordering of Scgb1a1 lineage-labeled cells colored by cluster assignment (right) according to diffusion pseudotime (DPT, left) order. Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CLDN4 (for DATPs, white), CC10 (G, green), AGER (H, for AT1 cell, green), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CLDN4+ DATPs in (G and H). Data are presented as mean ± SEM (n=5 mice for each genotype).
a) Schematic outline of the MAPPIT technique.figf1(a) Schematic outline of the MAPPIT technique. The indicated RAL mutants (baits) were fused to a leptin receptor fragment that is deficient in STAT3 recruitment sites. The RAL downstream effectors (preys) were tethered to a GP130 cytokine receptor fragment containing functional STAT3-docking sites. Association of bait and prey proteins restores a functional leptin receptor complex. On leptin stimulation, STAT3 activation can be monitored by a STAT3-responsive luciferase reporter. WT, wild type. (b) MAPPIT screen identified RAL-interacting proteins. A MAPPIT array covering close to 8,500 preys was screened with RALA-G23V as bait. Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1. (c) MAPPIT assay confirmed interaction between the RAL proteins and USP33. RALA-G23V or RALB-G23V baits were introduced into HEK293T cells together with the indicated preys. The results are expressed as a mean of normalized luciferase activity ± s.d (leptin-treated cells versus leptin-untreated cells) for three independent experiments. Statistics source data can be found in Supplementary Table S1. (d,e) USP33 interacts with both RALA and RALB. At 48 h post-transfection with tagged USP33 and RAL expression constructs, the indicated proteins were then immunoprecipitated with anti-Flag (M2) or anti-HA-agarose, followed by immunoblotting using anti-Flag or anti-HA antibodies. WCL, whole-cell lysate; IP, immunoprecipitates. (f) Endogenous USP33 interacts with RALB. Flag-RALB was immunoprecipitated using anti-Flag (M2) agarose and analysed for co-precipitation with endogenous USP33 using anti-USP33 antibody. Cells expressing USP33 shRNA were used as a negative control. (g) GTP binding is not required for interaction between USP33 and the RAL proteins. The GST-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody. (h) USP33 affects ubiquitylation of RALB, but not RALA. 6xHis-tagged ubiquitin and Flag-RAL-G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA. Ubiquitylated RAL proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALA or RALB. Uncropped images of blots are shown in Supplementary Fig. S7. (g) GTP binding is not required for interaction between USP33 and the RAL proteins. The USP33-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.
Aged cardiomyocytes activate senescence pathways but not a typical SASP.Figure 4Schematic illustrating CM isolation procedure. Real-time PCR gene expression analysis in isolated mouse CMs from C57BL/6 mice. Mean % of p21-positive CM nuclei from 3, 15, and 30 month old C57BL/6 mice by immunohistochemistry (IHC). Data are mean ± SEM of n=4 per age group. >100 CMs were quantified per age group. Mean % of 3 and 24 month old mouse CMs staining positive for SA-β-Gal in vivo with representative images above (blue - SA-β-Gal; green - troponin C; red - WGA). Black arrows indicate SA-β-Gal expression in a troponin C expressing CM. Statistical analysis performed using two-tailed t-test; *P<0.05. Data are mean ± SEM from the analysis of >500 CMs per mouse, 4 mice per age group. Scale bar 20µm. Mean % of SADS-positive CM nuclei from 3 and 30 month old mouse CMs positive for SADS in vivo, as detected by centromere-FISH. Data are mean ± SEM of n=4 per age group. >200 CMs per mouse were quantified. SASP heatmap: Pearson correlation clustered heatmap showing a curated list of known SASP genes (top panel) or a selection of secreted SASP proteins (bottom panel) in young (3 months) and old (20 months) mouse CMs (n=5 per age group). The colour intensity represents column Z-score, where red indicates highly and blue lowly expressed.
Figure 6A. Blue native PAGE analysis of RISP and CORE1 assembled into free cIII2 and supercomplexes (SCs) B-C. RISP per CORE1 ratio in (B) SC1 and (C) free cIII2. Spectrophotometric assay of (D) cI, (E) cII activity in isolated mitochondria at P200 (n=4-6/group) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g Spectrophotometric assay o (F) cIII activity in isolated mitochondria at P200 (n=4-6/group) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g G. CIV activity as measured using an oxygraph Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g H. n-Propyl gallate-sensitive AOX mediated cI and cII-linked state 3 respiration (n=7/group, Mann-Whitney U test) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g CI-linked (I state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) We used malate, pyruvate and glutamate to generate NADH for the cI Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g cI- and cII-linke state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) we added the CII substrate, succinate, to obtain CI&CII-linked respiration in presence of saturating ADP. Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g
SARS-CoV-2 Spike protein induces IL-1β secretion in macrophages from COVID-19 patients.Figure 1SDS PAGE of the recombinant SARS-CoV-2 spike protein (S-protein). SARS-CoV-2 spike binding assay of IgGs isolated from COVID-19 patients (n = 3; red circles) or SARS-CoV-2 naïve individuals (SC-naïve) (n = 2; blue circles). Experimental scheme: After PBMCs isolation, CD14+ cells were enriched by positive selection. Subsequently, 5x104 CD14+ cells/well were seeded and incubated in the presence of M-CSF for 5 days. Differentiated macrophages were stimulated with/without recombinant SARS-CoV-2 spike protein or lipopolysaccharide (LPS) for 4h. To activate IL-1β secretion nigericin or ATP was added for 2h. Finally, IL-1β secretion was quantified by ELISA. Quantification of IL-1β concentration (pg/ml) in the supernatant of primary macrophage cultures from COVID-19 patients (n = 44; red bars) or SC-naïve individuals (n = 24; blue bars) stimulated with LPS or S-protein (0.1 µg/ml). To activate IL-1β secretion nigericin was added for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used. TNF-α (pg/ml) secreted from macrophages derived from SC-naïve and COVID-19 patients as described above. Cells were stimulated with LPS (SC-naïve/COVID-19 n = 4), LPS with nigericin (SC-naïve n = 4; COVID-19 n = 6), S-protein (SC-naïve n = 5; COVID-19 n = 4), S-protein with nigericin (SC-naïve n = 3; COVID-19 n = 4) or left unstimulated (SC-naïve n = 3; COVID-19 n = 4). Significances shown in the figure are always in comparison to the unstimulated control.
Figure 1A. Predicated MLS localized at the C-terminus of GASZ and is enriched for hydrophobic amino acids that form a helical wheel. B. Subcellular fractionation and Western blots of 293T cells with ectopically expressed GASZ, NRF1, MVH, or MitoPLD. C. Localization of GFP fused with wild-type or mutant MLS of GASZ in HeLa cells. Mitochondrial localization was monitored by MitoTracker (Red) staining. Scale bars: 15 m.