recombinant pad4  (Sino Biological)

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: a Chemical structures of PAD-PF1, GSK147, and PAD-PF2. b PAD-PF1 inhibits PAD4-mediated citrullination of peptide substrate Atto655-(Ser-Arg-Gly-Ala) 3 in a fluorescence quenching (FQ) assay in the presence of 0.4 mM Ca 2+ . Data points and error bars represent the mean and SD of quadruplicate determinations of a representative experiment. IC 50 = 15.9 µM, pIC 50 = 4.80 (0.26), mean (SD), n = 29 biologically independent experiments. c – e Representative surface plasmon resonance (SPR) traces characterizing binding of respective ligands to Bap-tagged PAD4 captured on streptavidine sensor. The red line represents the fitted curve. c PAD-PF1. K D = 2.82 µM, pK D = 5.55 (0.12), mean (SD), n = 4 technical replicates. The highest concentration of PAD-PF1 tested was 10 µM, with five more 3-fold dilutions. d , GSK147. K D = 0.469 µM, pK D = 6.33 (0.016), mean (SD), n = 4 technical replicates. The highest concentration of GSK147 tested was 3.33 µM with five more 3-fold dilutions. e Same concentration series of GSK147 in the presence of 10 µM PAD-PF1. K D = 0.380 µM, pK D = 6.42 (0.071), mean (SD), n = 4 technical replicates. Source data are provided as a Source Data file.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Fluorescence, SPR Assay, Binding Assay, Concentration Assay

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: a Crystal structure of human full-length recombinant PAD4 simultaneously bound to GSK147 (orange carbon sticks and van der Waals surface) and PAD-PF1 (green carbon sticks and van der Waals surface). Protein backbone displayed as a ribbon diagram. b PAD4 binding pocket of GSK147 (orange carbon sticks). c PAD4 binding pocket of PAD-PF1 (green carbon sticks). In ( b , c ), side chains interacting with the small molecules are shown as white carbon sticks, hydrogen bonds are dotted lines, and water molecules are represented as red spheres. Key protein residues with hydrophobic interactions with the ligand are labeled in red.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Recombinant, Binding Assay, Labeling

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: a PAD-PF2 (open circles) inhibits PAD4-mediated citrullination of peptide substrate Atto655-(Ser-Arg-Gly-Ala) 3 employing a fluorescence quenching (FQ) assay in the presence of 0.4 mM Ca 2+ . Data points and error bars represent the mean and SD of three independent experiments in duplicate. IC 50 = 42.7 nM, pIC 50 = 7.37 (0.03), mean (SD), n = 3 independent experiments. The enantiomer of PAD-PF2 ( ent -PAD-PF2, open squares) was less potent. IC 50 = 26.0 µM, pIC 50 = 4.59 (0.45), mean (SD), n = 3 independent experiments. b PAD-PF2 induces a thermal stabilization of PAD2 (open circles, EC 50 = 2.04 µM) and PAD4 (open squares, EC 50 = 1.95 µM) protein. Individual data points shown for a duplicate determination. c – f Inhibition of the respective PAD isoform by PAD-PF2 determined using the GDH-enzyme coupled assay in the presence of 0.25 mM Ca 2+ . Individual data points shown for a duplicate determination along with the fitted IC 50 curves (line). c PAD1, IC 50 = 109 nM. d PAD2, IC 50 = 28.5 nM. e PAD3, IC 50 = 106 nM. f PAD4, IC 50 = 24.0 nM. g , h Calcium dependence of PAD2 or PAD4 inhibition by PAD-PF2. Inhibition was determined using the GDH enzyme-coupled assay. Data points from duplicate experiments performed at high (open squares) and low (open circles) Ca 2+ concentrations are plotted together, and the fitted IC 50 curves are shown (line). g The determined IC 50 values for PAD2 inhibition were 27.9 nM and 3.09 µM (0.25 and 1.5 mM Ca 2+ , respectively). h The determined IC 50 values for PAD4 inhibition were 20.1 nM and 694 nM (0.25 and 1.5 mM Ca 2+ , respectively). Source data are provided as a Source Data file.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Fluorescence, Inhibition

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: The method of Loewe was used to assess synergy between PAD-PF2 and GSK147 towards inhibition of PAD4. Serial dilutions of the inhibitors (0–3.6 μM) were tested in all combinations (two-by-two matrix) under the PAD4 GDH-coupled assay condition with 1.5 mM CaCl 2 . Plotted are the normalized doses of the two compounds that were observed to produce half-maximal activity. For circles, x = [GSK147]/GSK147 IC 50 in the absence of PAD-PF2, y = PAD-PF2 IC 50 /PAD-PF2 IC 50 in the absence of GSK147. For squares, x = GSK147 IC 50 /GSK147 IC 50 in the absence of PAD-PF2, y = [PAD-PF2]/PAD-PF2 IC 50 in the absence of GSK147. Isobole represented by the dotted line. Source data are provided as a Source Data file.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Inhibition, Activity Assay

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: a Crystal structure of human full-length recombinant PAD4 bound to PAD-PF2 (green carbon sticks). The disordered side chains of Asp369 and Ser370 were constructed using the program ADDS . b Crystal structure of human full-length recombinant PAD2 bound to PAD-PF2 (green carbon sticks). In a , b , side chains interacting with the small molecules are shown as white carbon sticks, hydrogen bonds are dotted lines, and water molecules are represented as red spheres. Key protein residues with hydrophobic interactions with the ligand are labeled in red.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Recombinant, Construct, Labeling

Journal: Redox Biology

Article Title: Inhibition of S100A8/A9 ameliorates neuroinflammation by blocking NET formation following traumatic brain injury ☆

doi: 10.1016/j.redox.2025.103532

Figure Lengend Snippet: PAD4 deficiency abolished the effects of S100A8/A9 on neurological functions and inflammatory/apoptotic protein levels after TBI . A Quantitative analysis of DNA in the supernatant from neutrophils in vitro treated with S100A8/A9. ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group. B, C Representative immunofluorescence staining NETs (H3cit, green) (B) and quantitative analyses of H3cit-positive cells (C) in neutrophils in vitro. Nuclei were stained with DAPI (blue). Scale bar = 50 μm ∗∗∗p < 0.001, n = 6 per group. D, E Representative immunofluorescence staining (D) of DCFH-DA (green) and quantitative analyses of fluorescence intensity of DCFH-DA (E) in neutrophils in vitro. Scale bar = 50 μm ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group. F Quantitative analyses of plasma DNA at 3 d after TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group. G, H mNSS test (G) and Rotarod test (H) at 3 d post-TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 8 per group. I–K pro-inflammation-associated mRNA levels were evaluated including CD86 (I) , IL-1β (J) , and IL-6 (K) . ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 8 per group. L, M anti-inflammation-associated mRNA levels were evaluated including CD206 (L) and Arginase-1 (M) . ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 8 per group. N Representative image of the FJC (green) staining in the cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. O Quantitative analyses of FJC-positive cells in the peri-contusional cortex at 3 d after TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group.

Article Snippet: Recombinant PAD4 adenovirus (Ad-PAD4; Adeno-CMV-Padi4-3∗flag-tagged SV40-EGFP, 4 × 10 10 plaque-forming-unit/mL) and control adenovirus (Ad-con, Adeno-CMV-3∗flag-tagged SV40-EGFP) were purchased from Genechem (Shanghai, China).

Techniques: In Vitro, Immunofluorescence, Staining, Fluorescence, Clinical Proteomics

Journal: Redox Biology

Article Title: Inhibition of S100A8/A9 ameliorates neuroinflammation by blocking NET formation following traumatic brain injury ☆

doi: 10.1016/j.redox.2025.103532

Figure Lengend Snippet: PAD4 overexpression abolished the neuroprotective effects of PAQ on neurological functions and inflammatory/apoptotic proteins levels after TBI . A Schematic image of adeno-PAD4-EGFP and overexpression of PAD4 in the cortex of the mouse. B, C mNSS test (B) and Rotarod test (C) on days 3 post-TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 8 per group. D Representative double immunofluorescence staining for Iba1 (red) and CD16/32 (green) in the contused cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. E Quantitative analyses of CD16/32-positive microglia/macrophage in the peri-contusional cortex at 3 d after TBI. ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group. F Representative double immunofluorescence staining for Iba1 (red) and Arginase-1 (green) in the contused cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. G Quantitative analyses of Arginase-positive microglia/macrophage in the peri-contusional cortex at 3 d after TBI. ∗∗∗p < 0.001, n = 6 per group. H Representative image of the FJC (green) staining in the cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. I Quantitative analyses of FJC-positive cells in the peri-contusional cortex at 3 d after TBI. ∗∗∗p < 0.001, n = 6 per group. J Representative image of the TUNEL (green) co-localization with neurons (NeuN, red) in the cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. K Quantitative analyses of TUNEL-positive neurons in the peri-contusional cortex at 3 d after TBI. ∗p < 0.05, ∗∗p < 0.01, n = 6 per group. L Representative Western blot bands and densitometric quantification of TNF-α, IL-1β, Arginase-1 , Bcl-2, Bax , and C-Caspase-3 after TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group.

Article Snippet: Recombinant PAD4 adenovirus (Ad-PAD4; Adeno-CMV-Padi4-3∗flag-tagged SV40-EGFP, 4 × 10 10 plaque-forming-unit/mL) and control adenovirus (Ad-con, Adeno-CMV-3∗flag-tagged SV40-EGFP) were purchased from Genechem (Shanghai, China).

Techniques: Over Expression, Double Immunofluorescence Staining, Staining, TUNEL Assay, Western Blot

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) Differential expression analysis of PTM enzymes in TAMs vs. normal macrophages from patients with breast cancer (GEO: GSE117970). (B) Human PADI4 mRNA expression in normal breast macrophages vs. in breast cancer TAMs examined in a bulk RNA sequencing (RNA-seq) dataset (GEO: GSE117970) (n = 4). (C) Differential expression analysis of PTM enzymes in TAMs vs. normal macrophages from breast-tumor-bearing mice (GEO: GSE212643). (D) Mouse Padi4 mRNA expression in normal breast macrophages vs. in breast cancer TAMs examined in a bulk RNA-seq dataset (GEO: GSE212643) (n = 3). (E) t-Distributed stochastic neighbor embedding (tSNE) plots generated from scRNA-seq data (GEO: GSE121521) showing distribution of macrophage-associated genes across peritoneal lavage subsets from mice. (F) Proportion of Padi4 + cells in each immune cell subset of the mouse peritoneal lavage (GEO: GSE121521). (G) Expression levels of Padi4 across immune cell subsets of the mouse peritoneal lavage (GEO: GSE121521). (H) Proportion of PADI4 + cells in each immune cell subset of peripheral blood mononuclear cells from peripheral blood mononuclear cells (PBMCs) from patients with colorectal cancer (GEO: GSE146771). Data are shown as mean ± SEM (B, D, and G). Unpaired two-tailed student’s t test (B and D). One-way ANOVA test (G). *p < 0.05, **p < 0.01, and ****p < 0.0001. scRNA-seq, single-cell RNA sequencing; ns, not significant.

Article Snippet: Recombinant anti-PADI4/PAD4 Rabbit mAb [EPR20706] , Abcam , Cat# ab214810.

Techniques: Expressing, RNA Sequencing Assay, Generated, 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: (A) Growth kinetics of subcutaneous MC38 murine colorectal cancer in Padi4 +/+ and Padi4 −/− mice (n = 6). (B) At endpoint, MC38 tumors from Padi4 +/+ and Padi4 −/− mice were excised (n = 6). (C and D) Percentages of IFNγ + (C) and IL-2 + (D) T cells from MC38-tumor-bearing Padi4 +/+ and Padi4 −/− mice (n = 5). (E) Growth kinetics of subcutaneous MC38 murine colorectal cancer in Padi4 fl/fl and Padi4 fl/fl LysM cre mice (n = 5). (F) At endpoint, MC38 tumors from Padi4 fl/fl and Padi4 fl/fl LysM cre mice were excised (n = 5). (G and H) Percentages of IFNγ + CD4 + (G) and IFNγ + CD8 + (H) T cells from MC38 tumors of Padi4 fl/fl and Padi4 fl/fl LysM cre mice (n = 5). (I and J) Percentages of TNFα + IFNγ + CD4 + (I) and TNFα + IFNγ + CD8 + (J) T cells from MC38 tumors of Padi4 fl/fl and Padi4 fl/fl LysM cre mice (n = 5). (K) Mouse IFNγ enzyme-linked immunosorbent spot (ELISpot) assay measuring IFNγ production in tumor-infiltrating T cells from Padi4 fl/fl and Padi4 fl/fl LysM cre MC38-bearing mice following stimulation with dead ultraviolet (UV)-irradiated MC38 tumor cells (n = 5–6). (L) Growth kinetics of subcutaneous Py8119 murine breast cancer in Padi4 fl/fl and Padi4 fl/fl LysM cre mice (n = 5). (M) At endpoint, Py8119 tumors from Padi4 fl/fl and Padi4 fl/fl LysM cre mice were excised (n = 5). (N) Lung nodule counts on the metastatic lungs excised from Padi4 fl/fl and Padi4 fl/fl LysM cre mice intravenously inoculated with B16F10 (n = 7). (O–R) Percentages of IFNγ + (O and P) and TNFα + (Q and R) tumor-infiltrated T cells from the lung metastasis of B16F10-bearing Padi4 fl/fl and Padi4 fl/fl LysM cre mice (n = 7). Data are shown as mean ± SEM (A, C–E, G–L, N, O, and Q). Unpaired two-tailed Student’s t test. *p < 0.05; **p < 0.01.

Article Snippet: Recombinant anti-PADI4/PAD4 Rabbit mAb [EPR20706] , Abcam , Cat# ab214810.

Techniques: ELISpot Assay, Enzyme-linked Immunospot, Irradiation, 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: (A) Representative histogram quantifying MHC class II protein expression in peritoneal and lung macrophages from Padi4 +/+ and Padi4 −/− mice. (B) Mean fluorescence intensity (MFI) of MHC class II expression on unchallenged primary peritoneal macrophages harvested from Padi4 +/+ and Padi4 −/− mice. Representative of nine independent experiments. (C) MFI of MHC class II expression on unchallenged primary lung macrophages harvested from Padi4 +/+ and Padi4 −/− mice. Representative of four independent experiments. (D) Quantitative polymerase chain reaction (qPCR) results showing MHC class II-coding and IFNγ-responsive gene expression in the peritoneal macrophages from healthy Padi4 +/+ vs. Padi4 −/− mice (n = 6/group, qPCR normalized to β-actin expression). (E) Fluorescence-activated cell sorting (FACS) analysis showing the MFI of MHC class II in Lyve1 + and Lyve1 – tumor macrophages from MC38-tumor-bearing Padi4 fl/fl vs. Padi4 fl/fl LysM cre mice (n = 5). (F) FACS analysis showing the MFI of MHC class II in Lyve1 + and Lyve1 – tumor macrophages from Py8119-tumor-bearing Padi4 fl/fl vs. Padi4 fl/fl LysM cre mice (n = 5). (G) FACS analysis showing the MFI of MHC class II in alveolar and interstitial lung tumor macrophages from B16F10-bearing Padi4 fl/fl vs. Padi4 fl/fl LysM cre mice (n = 7). (H) Gene set enrichment analysis (GSEA) of Padi4 high mouse peritoneal macrophages from scRNA-seq data of murine peritoneal lavage (GEO: GSE121521) (top). GSEA of PADI4 high human blood monocytes from scRNA-seq data of human PBMCs (GEO: GSE169246) (bottom). (I) Percentages of IFNγ + OT-II T cells cultured alone or with Padi4 +/+ or Padi4 −/− macrophages in the presence or absence of 10 5 UV-irradiated OVA + MC38 cells (n = 4). (J) Percentages of IL-2 + OT-II T cells cultured alone or with Padi4 +/+ or Padi4 −/− macrophages in the presence or absence of 10 5 UV-irradiated OVA + MC38 cells (n = 4). Data are shown as mean ± SEM (B–G, I, and J). Unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, and ***p < 0.001. MFI, mean fluorescence intensity; ns, not significant.

Article Snippet: Recombinant anti-PADI4/PAD4 Rabbit mAb [EPR20706] , Abcam , Cat# ab214810.

Techniques: Expressing, Fluorescence, Real-time Polymerase Chain Reaction, FACS, Cell Culture, Irradiation, 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: (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: Recombinant anti-PADI4/PAD4 Rabbit mAb [EPR20706] , Abcam , Cat# ab214810.

Techniques: Ex Vivo, Immunoprecipitation, 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: Recombinant anti-PADI4/PAD4 Rabbit mAb [EPR20706] , Abcam , Cat# ab214810.

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: Recombinant anti-PADI4/PAD4 Rabbit mAb [EPR20706] , Abcam , Cat# ab214810.

Techniques: Isolation, Flow Cytometry, Derivative Assay, Expressing, 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: Recombinant anti-PADI4/PAD4 Rabbit mAb [EPR20706] , Abcam , Cat# ab214810.

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, Expressing, Transgenic Assay, CRISPR, Software