Journal: eLife

Article Title: Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

doi: 10.7554/eLife.90683

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Article Snippet: Antibody , Goat anti-mouse IgG, Light chain specific (HRP) , Jackson ImmunoResearch Laboratories, Inc , Cat# 115-035-174; RRID: AB_2338512 , (1:5000).

Techniques: Recombinant, Plasmid Preparation, CRISPR, Knock-Out, Protease Inhibitor, Staining, Reverse Transcription, Mass Spectrometry, Transfection, Gene Knockout, Negative Control, Cell Culture, Plex Assay, Bicinchoninic Acid Protein Assay, Western Blot, Sensitive Assay, Cell Viability Assay, Software, Luminex, Real-time Polymerase Chain Reaction, Imaging

Journal: eLife

Article Title: eIF2B activator prevents neurological defects caused by a chronic integrated stress response

doi: 10.7554/eLife.42940

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Article Snippet: Antibody , Rabbit monoclonal anti-phospho- 4EBP1 , Cell Signaling Technology , #2855 (RRID: AB_560835 ) , Western Blot (1:1000 dilution).

Techniques: Mutagenesis, Synthesized, Quantigene Plex 2.0 Assay, Luciferase, Western Blot, Electron Microscopy, Staining

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, Identification of SERPINB1 as a binding partner of caspase-4 CARD (aa 2–124) by yeast two-hybrid screening. For yeast co-transformation assays, full-length and truncated forms of caspase-1/–4/–5 were co-transformed with the SERPINB1 carboxy-terminal region (aa 330–379) to yeast for growth on two dropout (DO) or four dropout containing X-α-Gal plates. b, Specific interaction between SERPINB1 and inflammatory caspase family members. GST–SERPINB1–330–379 and hemagglutinin (HA)-tagged CARD-containing caspases were transfected into 293 T cells, and whole-cell extracts (WCEs) were subjected to GST pulldown (GST-PD), followed by immunoblotting (IB) using anti-HA or anti-GST. c, Endogenous interaction between caspase-1/–4 and SERPINB1. THP1 WCEs were subjected to co-immunoprecipitation (IP) with control anti-HA or anti-caspase-1. U937 WCEs were subjected to co-immunoprecipitation with control anti-HA or anti-caspase-4. mAb and rAb denote mouse and rabbit antibodies, respectively. HC, heavy chain. d, Specific binding of caspase-4 to SERPINB1. HA-tagged caspase-4 and Flag-tagged serpin family members were transfected into 293 T cells, and WCEs were subjected to co-immunoprecipitation with anti-Flag, followed by immunoblotting using anti-HA or anti-Flag. e, Interaction of enzymatically inactive caspase mutants with SERPINB1. HA-tagged caspase-1 or caspase-4 wild-type or enzymatically inactive mutants and Flag-tagged SERPINB1 were transfected into 293 T cells, and WCEs were subjected to co-immunoprecipitation with anti-HA, followed by immunoblotting with anti-Flag or anti-HA. Data in a–e are representative of two independent experiments.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Binding Assay, Two Hybrid Screening, Transformation Assay, Transfection, Western Blot, Immunoprecipitation, Control

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, Schematic diagram of SERPINB1 truncation and alanine-substitution mutant constructs. The relative binding intensity is indicated as + ++ > ++ > + > (+ ) > −. b,c, GST-pulldown assay of SERPINB1 truncation mutants binding to caspase-1 and caspase-4. GST-SERPINB1 truncated forms and caspase-1-C285A–HA or caspase-4-C258A–HA were transfected into 293 T cells, and WCEs were subjected to GST pulldown, followed by immunoblotting using anti-HA or anti-GST. d, GST-pulldown assay of SERPINB1 alanine-substitution mutants binding to caspase-4. GST-SERPINB1 truncated or alanine-substituted forms and caspase-4-C258A–HA were transfected into 293 T cells, and WCEs were subjected to GST pulldown, followed by immunoblotting using anti-HA or anti-GST. Data in b–d are representative of two independent experiments.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Mutagenesis, Construct, Binding Assay, GST Pulldown Assay, Transfection, Western Blot

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, IL-1β secretion after SERPINB1 depletion. THP1 cells were transduced with scrambled or SERPINB1-specific shRNA (shSERPINB1) lentivirus for 48 h and were primed with LPS (1 μg ml−1) for 12 h. b, Immunoblots of the secreted p17 form of IL-1β after SERPINB1 depletion. c, Effects of deletion of the gene encoding caspase-1 or caspase-4 on SERPINB1-depletion-induced IL-1β secretion. d, Immunoblots of the cleaved p20 form of caspase-1 and processed p17 form of IL-1β in THP1-sgNT or THP1-sgCASP1 cells after SERPINB1 depletion. e,f, Effects of knockdown of genes encoding neutrophil elastase (shNE), proteinase-3 (shPRTN3), cathepsin G (shCG), ASC (shASC) or NLRP3 (shNLRP3) on SERPINB1-depletion-induced IL-1β secretion. g, Effects of caspase inhibitors on SERPINB1-depletion-induced cell death. SYTOX Green–based cell death was measured without or with LPS priming. h, Transmission electron microscopic images of U937 cells after SERPINB1 depletion. For a positive control, cells were electroporated with 1 μg LPS. Large vacuoles (filled triangles), disrupted nucleus (asterisks), swollen mitochondria (arrows). Scale bars, 1 μm. N, nucleus; V, phagocytic vacuole; M, mitochondria. Data in b,d,g,h are representative of two independent experiments. Whole-cell lysates (WCLs) and supernatants (SUPs) in b,d were immunoblotted with indicated antibodies. Data are presented as mean ± s.e.m. from n = 3 independent experiments in a,e,f and from n = 5 independent experiments in c, and as box and whiskers (min to max) from n = 3 technical replicates in g. P values were determined by one-way analysis of variance (ANOVA) with Dunnett’s comparison relative to scramble in a and by two-way ANOVA with Bonferroni’s comparison relative to non-targeting control (sgNT) in c, to scramble in e,f, or to dimethylsulfoxide (DMSO) in g. NS, not significant.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Transduction, shRNA, Western Blot, Knockdown, Transmission Assay, Positive Control, Comparison, Control

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, The RCL and CBM sequence alignment of human SERPINB1 and mouse SERPINB1a, SERPINB1b and SERPINB1c. The conserved P2–P1–P1 residues in the RCL sequence are indicated by orange font, and the CBM sequence is shown in red font. The conserved 13 aa in the CBM sequence are indicated in the red-dotted box. Specified amino acid numbers are based on human SERPINB1. b, Verification of Serpinb1-targeting shRNA’s silencing efficiency by qRT-PCR in DC2.4 cells. mRNA expression was normalized to 18S, and fold change was calculated relative to scramble. c, Immunoblots of cleaved p10 subunit of caspase-1 in DC2.4 cells after Serpinb1a, Serpinb1b and/or Serpinb1c depletion. WCLs and SUPs were obtained at 48 h post-transduction and were immunoblotted with indicated antibodies. d, Caspase-1 activation in Serpinb1a−/− pPMNs. FLICA-positive staining was analyzed by flow cytometry. e,f, IL-1β secretion and cell death after Serpinb1 depletion in BMDMs. Wild-type (WT) and Casp1−/−Casp11−/− BMDMs were transduced with scrambled or pan-Serpinb1 shRNA lentivirus. Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA), and cell viability was determined by ATP-based assay. Data in c are representative of two independent experiments. Data are presented as mean ± s.e.m. from n = 3 independent experiments in b,f and from n = 7 per group, pooled from two independent experiments in d, and as box and whiskers (min to max) from n = 6 pooled from three independent experiments in e. P values were determined by one-way ANOVA with Dunnett’s comparison relative to scramble in b, by an two-tailed unpaired t-test in d and by two-way ANOVA with Bonferroni’s comparison relative to scramble in e,f.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Transduction, Activation Assay, Staining, Flow Cytometry, shRNA, Enzyme-linked Immunosorbent Assay, ATP Assay, Comparison, Two Tailed Test

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, Survival of wild-type (WT) and Serpinb1a−/− mice (n = 12 per group) intraperitoneally challenged with 20 mg kg−1 LPS. b,c, Plasma IL-1β and liver and spleen Il6 and Cox2 mRNAs of wild-type and Serpinb1a−/− mice (n = 6 for the PBS group and n = 11 for the LPS group). Serum and organs were collected at 3 h post-injection. IL-1β concentrations were determined by ELISA. mRNA expression was normalized to 18 S, and fold change was calculated relative to the PBS-treated wild-type. d, Survival of wild-type, Serpinb1a−/−, Casp1−/−Casp11−/− and Serpinb1a−/−Casp1−/−Casp11−/− mice (n = 5 per group) intravenously infected with 1.2 × 107 to 1.5 × 107 colony-forming units of HUMC1. e, Plasma cytokine concentrations of wild-type and Serpinb1a−/− mice (n = 3–4 per group at 0 hpi, n = 5 per group at 6 hpi, n = 7 per group at 20 hpi). Cytokine concentrations were quantified using a Bio-Plex assay. f, Blood bacterial burden of wild-type and Serpinb1a−/− mice (n = 10 for wild-type or Serpinb1a−/− at 6 hpi, n = 7 for wild-type or Serpinb1a−/− at 20 hpi, n = 5 for Casp1−/−Casp11−/− or Serpinb1a−/−Casp1−/−Casp11−/− at 6 or 20 hpi). Data are presented as Kaplan–Meier plot in a,d, as mean ± s.e.m. in b,c,f, and as box and whiskers (min to max) with line at median in e. P values were determined by log-rank test in a, by two-way ANOVA with Bonferroni’s comparison relative to wild-type in b,c,e, and by one-way ANOVA with Dunnett’s comparison relative to wild-type in f. NS, not significant.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Clinical Proteomics, Injection, Enzyme-linked Immunosorbent Assay, Expressing, Infection, Plex Assay, Comparison

Journal: Nature immunology

Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

doi: 10.1038/s41590-018-0303-z

Figure Lengend Snippet: a, Increased oligomerization of endogenous caspase-1/–4 after SERPINB1 depletion. Scramble or shSERPINB1 lentivirus–infected THP1 cells were pre-incubated with z-VAD-FMK and LPS for 6 h before DSS crosslinking. Crosslinked cells were lysed, and WCEs were immunoblotted with indicated antibodies. Square bracket (right margin) indicates oligomerized caspase-1 or caspase-4. b, Size-exclusion chromatography assay of caspase-1 CARD oligomerization. MBP-1CARD-SUMO was incubated with or without SUMO-SERPINB1 at 4 °C for 12 h and subjected to TEV cleavage. The indicated samples were analyzed by size-exclusion columns. SDS-PAGE of each fraction was stained with Coomassie blue. The red-dotted box indicates the fractions containing 1CARD-SUMO proteins. c, SERPINB1-mediated inhibition of caspase-1 CARD oligomerization. MBP-1CARD-SUMO (100 nM) was incubated with or without SERPINB1 (100 nM) at 4 °C for 12 h and was subjected to TEV cleavage for 2 h, followed by BS3 crosslinking. Crosslinked proteins were immunoblotted with indicated antibodies. Relative density was calculated by Image Lab Software. d,e, Fluorescence polarization assay of caspase-1 CARD oligomerization. SERPINB1 was incubated with FITC-conjugated MBP-1CARD-SUMO or together with MBP-ASC at 4 °C for 12 h and was subjected to TEV cleavage. The fluorescence polarization signals were collected at the indicated time point with an Envision plate reader. Data in a–e are representative of two independent experiments. Data in d,e are presented as mean ± s.e.m. from n = 3 technical replicates.

Article Snippet: Primary antibodies include antibodies to human SERPINB1 (3B4, Origene), human caspase-1 (14F468, Santa Cruz), human caspase-1 ( {"type":"entrez-protein","attrs":{"text":"EPR16883","term_id":"523382955","term_text":"EPR16883"}} EPR16883 , Abcam), human caspase-1 p20 (Bally-1, Adipogen), human caspase-1 CARD (A-19, Santa Cruz), mouse caspase-1 (M-20, Santa Cruz), human caspase-4 (4B9, Santa Cruz), caspase-2 (611022, BD), caspase-3 (8G10, Cell Signaling), IL-1β (2002, Cell Signaling), neutrophil elastase (C-17, Santa Cruz), proteinase-3 (684042, R&D Systems), cathepsin G (N-19, Santa Cruz), NLRP3 (Cryo-2, Adipogen), ASC (F-9, Santa Cruz), SMT3 (y-84, Santa Cruz), His (H-15, Santa Cruz), GST (B-14, Santa Cruz), Actin (C4, Santa Cruz), rabbit-Flag (F7425, Sigma), mouse-Flag (F1804, Sigma), rabbit-HA (PRB-101P, Covance), mouse-HA (16B12, BioLegend), mouse IgG-HRP (7076, Cell Signaling), rabbit IgG-HRP (7074, Cell Signaling) and goat IgG-HRP (sc-2020, Santa Cruz).

Techniques: Infection, Incubation, Size-exclusion Chromatography, SDS Page, Staining, Inhibition, Software, Fluorescence