phosphor stat1 tyr107  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: PAD4 controls tumor immunity via restraining the MHC class II machinery in macrophages

doi: 10.1016/j.celrep.2024.113942

Figure Lengend Snippet: (A) Tim-4 + peritoneal macrophages from Padi4 +/+ and Padi4 −/− mice were stimulated with 10 ng/mL IFNγ ex vivo for 1 h. Whole-cell lysates from Padi4 +/+ vs. Padi4 −/− Tim-4 + macrophages were subjected to immunoprecipitation with anti-STAT1 or control immunoglobulin G (IgG). The immunoprecipitant was probed with anti-PAD4. (B) Padi4 +/+ and Padi4 −/− primary mouse Tim-4 + -enriched peritoneal macrophages were stimulated with 10 ng/mL IFNγ ex vivo for 1 h. STAT1 citrullination was detected via streptavidin pull-down of citrulline-labeled proteins and probed with anti-STAT1. (C and D) 10 ng/mL IFNγ (C) or 1 μg/mL LPS (D) stimulation of Padi4 +/+ vs. Padi4 −/− splenocytes for 1 h followed by the detection of citrullinated STAT1. (E) Treatment of HL60 cells with 10 ng/mL IFNγ and 10 μM GSK484 or DMSO followed by the detection of citrullinated STAT1. (F) The in vitro citrullination assay performed with recombinant human PAD4 (0.5 μg) and recombinant human STAT1 (0.5 μg) proteins supplemented with 2 mM CaCl 2 and HEPES. (G) High-resolution precursor ion (MS1) isotopic envelopes of the R121 peptide of citrullinated STAT1. (H) MS2 fragmentation spectra originating from the same precursor ion. Observed b and y ions are indicated. Presence of unmodified b 6 and modified b 7 ions suggests that R121 is citrullinated. The resulting m/z of 826.43 due to the modified b 7 ions is indicated in red. (I) Protein Prospector results revealing the predicted m/z at the non-citrullinated vs. citrullinated R121 in the ILENAQRNQAQS peptide. m/z , mass-to-charge ratio.

Article Snippet: The primary antibodies against mouse PADI4 (1:1000, Abcam, ab214810), STAT1 (1:1000, CST, 9172), phosphor-STAT1 (Tyr107) (1:1000, CST, 9167), PIAS1 (1:1000, CST, 3350), β-actin (1:1000, CST, 3700), MHC-II (1:1000, Abcam, ab55152 or ab180779), CIITA (1:500, Abcam, ab70060) and HLA-DR (1:1000, Abcam, ab118347) were used.

Techniques: Ex Vivo, Immunoprecipitation, Control, Labeling, In Vitro, Recombinant, Modification

Journal: Cell reports

Article Title: PAD4 controls tumor immunity via restraining the MHC class II machinery in macrophages

doi: 10.1016/j.celrep.2024.113942

Figure Lengend Snippet: (A) Padi4 +/+ and Padi4 −/− bone marrow-derived macrophages were generated, and proteins were lysed and processed to detect STAT1 citrullination and for the co-immunoprecipitation with anti-PIAS1. (B) Peritoneal macrophages were harvested from Padi4 +/+ and Padi4 −/− mice and stimulated with 10 ng/mL IFNγ for 1 h. Proteins were lysed and processed for the co-immunoprecipitation with anti-PIAS1. (C) HL60 cells were treated with 1 μg/mL LPS for 1 h with or without GSK484, and proteins were lysed and processed for the co-immunoprecipitation with anti-PIAS1. (D) HL60 cells were treated with 10 ng/mL IFNγ for 1 h with or without GSK484, and proteins were lysed and processed for the co-immunoprecipitation with anti-PIAS1. (E) A mutant STAT1 HEK293T cell line in which the R121 was converted into K121 was generated. Cells were treated with 10 ng/mL IFNγ for 1 h, and proteins were lysed and processed to detect HLA-DR levels and for the co-immunoprecipitation with anti-PIAS1. (F) Chromatin immunoprecipitation was performed on DNA extracted from IFNγ-treated Padi4 +/+ and Padi4 −/− mouse splenocytes. qPCR primers for the detection of STAT1 at multiple IFNγ-responsive genomic regions in the CIITA gene were designed. Data are shown as mean ± SEM. n = 3–4. One-tailed Mann-Whitney U test. *p < 0.05.

Article Snippet: The primary antibodies against mouse PADI4 (1:1000, Abcam, ab214810), STAT1 (1:1000, CST, 9172), phosphor-STAT1 (Tyr107) (1:1000, CST, 9167), PIAS1 (1:1000, CST, 3350), β-actin (1:1000, CST, 3700), MHC-II (1:1000, Abcam, ab55152 or ab180779), CIITA (1:500, Abcam, ab70060) and HLA-DR (1:1000, Abcam, ab118347) were used.

Techniques: Derivative Assay, Generated, Immunoprecipitation, Mutagenesis, Chromatin Immunoprecipitation, One-tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: PAD4 controls tumor immunity via restraining the MHC class II machinery in macrophages

doi: 10.1016/j.celrep.2024.113942

Figure Lengend Snippet: (A) Primary human ovarian cancer mononuclear cells were isolated from patient tumors, treated with 10 ng/mL IFNγ and 10 μM GSK484 or DMSO, and then processed to detect CD45 + CD14 + HLA-DR levels via flow cytometry (n = 6). (B) Primary human macrophages were enriched and derived from PBMCs of blood buffy coats and treated with 10 ng/mL IFNγ and 10 μM GSK484 or DMSO. Proteins were lysed and processed to detect STAT1 citrullination and HLA-DR levels (n = 2). (C) CIITA and HLA-DRA expression in PAD4-deficient ( PADI4 low ) vs. PAD4-expressing ( PADI4 high ) macrophages in patients with TNBC. (D) GSEA was conducted on PADI4 high macrophages, and the normalized enrichment scores (NESs) were assessed for the response to IFNγ and antigen presentation via MHC class II pathways. (E) Pearson correlations were conducted between TAM PADI4 expression and the expression effector CD4 + T cell genes including TBX21 and IL12RB2 in patients with TNBC. (F) Macrophages were isolated from the total CD45 + population of sequenced single cells from patients with TNBC treated with anti-PD-L1 monoclonal antibody (mAb; GEO: GSE169246) (left). CD33 + TAMs were further filtered from total responder (R) and non-responder (NR) macrophages, and PADI4 expression was assessed between R and NR patients with TNBC (n = 5 responders, n = 6 non-responders) (right). (G) MC38 tumor progression in wild-type mice treated with or without 4 mg/kg GSK484 or 100 μg anti-PD-L1 mAb treatment (n = 5/group). Data are shown as mean ± SEM (C, F, and G). Paired two-tailed Student’s t test (A). Unpaired two-tailed Student’s t test (C, F, and G). *p < 0.05, **p < 0.01, and ****p < 0.0001.

Article Snippet: The primary antibodies against mouse PADI4 (1:1000, Abcam, ab214810), STAT1 (1:1000, CST, 9172), phosphor-STAT1 (Tyr107) (1:1000, CST, 9167), PIAS1 (1:1000, CST, 3350), β-actin (1:1000, CST, 3700), MHC-II (1:1000, Abcam, ab55152 or ab180779), CIITA (1:500, Abcam, ab70060) and HLA-DR (1:1000, Abcam, ab118347) were used.

Techniques: Isolation, Flow Cytometry, Derivative Assay, Expressing, Immunopeptidomics, Two Tailed Test

Journal: Cell reports

Article Title: PAD4 controls tumor immunity via restraining the MHC class II machinery in macrophages

doi: 10.1016/j.celrep.2024.113942

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The primary antibodies against mouse PADI4 (1:1000, Abcam, ab214810), STAT1 (1:1000, CST, 9172), phosphor-STAT1 (Tyr107) (1:1000, CST, 9167), PIAS1 (1:1000, CST, 3350), β-actin (1:1000, CST, 3700), MHC-II (1:1000, Abcam, ab55152 or ab180779), CIITA (1:500, Abcam, ab70060) and HLA-DR (1:1000, Abcam, ab118347) were used.

Techniques: Recombinant, Plasmid Preparation, Lysis, Protease Inhibitor, SYBR Green Assay, Western Blot, Chromatin Immunoprecipitation, Magnetic Beads, Mutagenesis, Cell Isolation, Isolation, Transfection, Enzyme-linked Immunospot, Mass Spectrometry, Gene Expression, Transgenic Assay, CRISPR, Software