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rabbit anti mouse pad4 (Proteintech)

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Proteintech rabbit anti mouse pad4

PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and <t>PAD4</t> (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Rabbit Anti Mouse Pad4, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti mouse pad4 - by Bioz Stars, 2026-05

94/100 stars

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1) Product Images from "Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response"

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

Journal: iScience

doi: 10.1016/j.isci.2026.115038

Figure Legend Snippet: PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Techniques Used: Expressing, In Vivo, In Vitro, Infection, Quantitative RT-PCR, Variant Assay, Nucleic Acid Electrophoresis, Control

SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Figure Legend Snippet: SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Techniques Used: Infection, Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Variant Assay, Multiplex Assay, Immunofluorescence, Staining, Control, Software, Incubation, Liquid Chromatography with Mass Spectroscopy

PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.

Figure Legend Snippet: PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.

Techniques Used: Inhibition, Infection, Quantitative RT-PCR, Western Blot, Expressing, Quantitative Proteomics

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Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Expressing, In Vivo, In Vitro, Infection, Quantitative RT-PCR, Variant Assay, Nucleic Acid Electrophoresis, Control

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Techniques: Infection, Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Variant Assay, Multiplex Assay, Immunofluorescence, Staining, Control, Software, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Western Blot, Expressing, Quantitative Proteomics

ACOD1/ITA inhibits the formation of NETs to attenuate airway inflammation in corticosteroid-resistant asthma. (A) The concentration of dsDNA in BMDNs stimulated with DEP and treated with ITA. (B) Representative immunofluorescence staining of CitH3 and MPO in BMDNs stimulated with DEP and treated with ITA. Scale bar, 25 μm. (C) The concentration of dsDNA in BALF of HDM/DEP-induced asthma with or without treatment of ITA. (D) Western blot analysis of CitH3 in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. (E) Representative immunofluorescence staining of CitH3 and MPO in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. The arrows indicate the colocalization of CitH3 and MPO. (F) The concentration of dsDNA in BALF of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (G) Western blot analysis of CitH3 in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (H) Representative immunofluorescence staining of CitH3 and MPO in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. The arrows indicate the colocalization of CitH3 and MPO. (I) Western blot analysis of PAD4 in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. (J) Immunohistochemical staining of PAD4 in lung tissue of HDM/DEP mice and treated with or without ITA. Scale bar, 20μm. (K) Western blot analysis of PAD4 in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (L) Immunohistochemical staining of PAD4 in lung tissue of HDM/DEP mice in WT or Acod1 -/- mice. Scale bar, 20μm. (M) Human blood samples were collected. Plasma, PBMCs, and neutrophils were isolated for further study. (N) The concentration of dsDNA in supernatant of PBNs stimulated with DEP for different time points and treated with ITA. *** P < 0.001, compared with the control group. (O) The concentration of dsDNA in plasma of healthy control, mild-to-moderate asthma patients, and severe asthma patients. (P) Correlation analysis of dsDNA and lung function index FEV 1 %pred, FEV 1 , and FEV 1 /FVC. (Q) HDM/DEP-induced asthmatic mice were established. NETs inhibitor DNase I was administered 1 hour before HDM and DEP challenge. (R) Representative H&E and PAS staining pictures of lung tissues (n = 5 mice per group). Scale bar, 100 μm. (S) Total cell, neutrophil, and eosinophil counts in BALF measured by flow cytometry (n = 5 mice per group). (T-U) The percentage of IL-4 + (Th2) and IL-17A + (Th17) cells in CD4 + T cells (gated on Live, CD45 + CD3 + CD4 + ) measured using flow cytometry (n = 4-5 mice per group). (V) Levels of inflammatory cytokines in BALF measured by using a cytometric bead array (n = 5 mice per group). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. CitH3, citrulline histone H3; dsDNA, double-stranded DNA; i.t., intratracheally; i.p., intraperitoneally; HDM, house dust mite; DEP, diesel exhaust particles; BALF, bronchoalveolar lavage fluid; BMDNs, bone marrow-derived neutrophils; ITA, itaconate; ACOD1, aconitate decarboxylase 1; PBMCs, peripheral blood mononuclear cells; PBNs, peripheral blood neutrophils; PAD4, peptidylarginine deiminase 4; NETs, neutrophil extracellular traps.

Journal: International Journal of Biological Sciences

Article Title: Itaconate Modulates Neutrophil Homeostasis to Ameliorate Airway Inflammation in Diesel Exhaust Particles-exacerbated Asthma via Inhibiting NETs Formation

doi: 10.7150/ijbs.124927

Figure Lengend Snippet: ACOD1/ITA inhibits the formation of NETs to attenuate airway inflammation in corticosteroid-resistant asthma. (A) The concentration of dsDNA in BMDNs stimulated with DEP and treated with ITA. (B) Representative immunofluorescence staining of CitH3 and MPO in BMDNs stimulated with DEP and treated with ITA. Scale bar, 25 μm. (C) The concentration of dsDNA in BALF of HDM/DEP-induced asthma with or without treatment of ITA. (D) Western blot analysis of CitH3 in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. (E) Representative immunofluorescence staining of CitH3 and MPO in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. The arrows indicate the colocalization of CitH3 and MPO. (F) The concentration of dsDNA in BALF of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (G) Western blot analysis of CitH3 in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (H) Representative immunofluorescence staining of CitH3 and MPO in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. The arrows indicate the colocalization of CitH3 and MPO. (I) Western blot analysis of PAD4 in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. (J) Immunohistochemical staining of PAD4 in lung tissue of HDM/DEP mice and treated with or without ITA. Scale bar, 20μm. (K) Western blot analysis of PAD4 in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (L) Immunohistochemical staining of PAD4 in lung tissue of HDM/DEP mice in WT or Acod1 -/- mice. Scale bar, 20μm. (M) Human blood samples were collected. Plasma, PBMCs, and neutrophils were isolated for further study. (N) The concentration of dsDNA in supernatant of PBNs stimulated with DEP for different time points and treated with ITA. *** P < 0.001, compared with the control group. (O) The concentration of dsDNA in plasma of healthy control, mild-to-moderate asthma patients, and severe asthma patients. (P) Correlation analysis of dsDNA and lung function index FEV 1 %pred, FEV 1 , and FEV 1 /FVC. (Q) HDM/DEP-induced asthmatic mice were established. NETs inhibitor DNase I was administered 1 hour before HDM and DEP challenge. (R) Representative H&E and PAS staining pictures of lung tissues (n = 5 mice per group). Scale bar, 100 μm. (S) Total cell, neutrophil, and eosinophil counts in BALF measured by flow cytometry (n = 5 mice per group). (T-U) The percentage of IL-4 + (Th2) and IL-17A + (Th17) cells in CD4 + T cells (gated on Live, CD45 + CD3 + CD4 + ) measured using flow cytometry (n = 4-5 mice per group). (V) Levels of inflammatory cytokines in BALF measured by using a cytometric bead array (n = 5 mice per group). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. CitH3, citrulline histone H3; dsDNA, double-stranded DNA; i.t., intratracheally; i.p., intraperitoneally; HDM, house dust mite; DEP, diesel exhaust particles; BALF, bronchoalveolar lavage fluid; BMDNs, bone marrow-derived neutrophils; ITA, itaconate; ACOD1, aconitate decarboxylase 1; PBMCs, peripheral blood mononuclear cells; PBNs, peripheral blood neutrophils; PAD4, peptidylarginine deiminase 4; NETs, neutrophil extracellular traps.

Article Snippet: Immunohistochemical analysis was performed using a rabbit anti-peptidyl arginine deiminase 4 (PAD4) antibody (Proteintech, USA) to evaluate the expression levels of PAD4.

Techniques: Concentration Assay, Immunofluorescence, Staining, Western Blot, Immunohistochemical staining, Clinical Proteomics, Isolation, Control, Flow Cytometry, Derivative Assay

Radiation upregulates PAD4 mRNA and protein expression in neutrophils. BMDNs were exposed to 10 Gy radiation. (A) PAD4 mRNA expression levels were assessed 4 h after irradiation via reverse transcription-quantitative PCR. PAD4 protein expression levels were evaluated by western blotting 20 h after irradiation. (B) Representative PAD4 western blot and (C) quantitative bar diagrams are shown. Groups were compared using an unpaired two-tailed Student's t-test. Data are expressed as the mean ± SEM (n=6/group). * P<0.05 and *** P<0.001. Cont, control; Irrad, irradiation; NET, neutrophil extracellular traps; BMDNs, bone marrow-derived neutrophils; PAD4, peptidyl arginine deiminase 4.

Journal: International Journal of Molecular Medicine

Article Title: High-dose X-ray irradiation induces NETosis via the eCIRP/TREM-1 axis in mouse neutrophils

doi: 10.3892/ijmm.2025.5598

Figure Lengend Snippet: Radiation upregulates PAD4 mRNA and protein expression in neutrophils. BMDNs were exposed to 10 Gy radiation. (A) PAD4 mRNA expression levels were assessed 4 h after irradiation via reverse transcription-quantitative PCR. PAD4 protein expression levels were evaluated by western blotting 20 h after irradiation. (B) Representative PAD4 western blot and (C) quantitative bar diagrams are shown. Groups were compared using an unpaired two-tailed Student's t-test. Data are expressed as the mean ± SEM (n=6/group). * P<0.05 and *** P<0.001. Cont, control; Irrad, irradiation; NET, neutrophil extracellular traps; BMDNs, bone marrow-derived neutrophils; PAD4, peptidyl arginine deiminase 4.

Article Snippet: After blocking with 0.1% casein in TBS for 1 h at room temperature, each membrane was incubated overnight at 4°C with primary antibodies of rabbit anti-PAD4 antibody (1:1,000; cat. no. 17373-1-AP; Proteintech Group, Inc.) and mouse anti-β-actin antibody (1:5,000; cat. no. A5441; MilliporeSigma).

Techniques: Expressing, Irradiation, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test, Control, Derivative Assay

Exposure to ionizing radiation induces NETs via eCIRP/TREM-1 axis. Radiation-induced eCIRP upregulates and activates TREM-1 on neutrophils, resulting in increased expression of PAD4 and NET formation, causing neutropenia. NETs, neutrophil extracellular traps; PAD4, peptidyl arginine deiminase 4; eCIRP, extracellular cold-inducible RNA-binding protein; TREM-1, triggering receptor expressed on myeloid cells-1; MPO, myeloperoxidase; cit, citrulline; CitH3, citrullinated H3. The schema was created in BioRender. Murao, A. (2025) https://BioRender.com/qhaiby0

Journal: International Journal of Molecular Medicine

Article Title: High-dose X-ray irradiation induces NETosis via the eCIRP/TREM-1 axis in mouse neutrophils

doi: 10.3892/ijmm.2025.5598

Figure Lengend Snippet: Exposure to ionizing radiation induces NETs via eCIRP/TREM-1 axis. Radiation-induced eCIRP upregulates and activates TREM-1 on neutrophils, resulting in increased expression of PAD4 and NET formation, causing neutropenia. NETs, neutrophil extracellular traps; PAD4, peptidyl arginine deiminase 4; eCIRP, extracellular cold-inducible RNA-binding protein; TREM-1, triggering receptor expressed on myeloid cells-1; MPO, myeloperoxidase; cit, citrulline; CitH3, citrullinated H3. The schema was created in BioRender. Murao, A. (2025) https://BioRender.com/qhaiby0

Techniques: Expressing, RNA Binding Assay

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