rabbit polyclonal anti cxcl5  (Abmart Inc)

Journal: Particle and Fibre Toxicology

Article Title: Inflammogenic effect of polyacrylic acid in rat lung following intratracheal instillation

doi: 10.1186/s12989-022-00448-z

Figure Lengend Snippet: Analysis of cytokines in BALF and HO-1 in lung tissue following intratracheal instillation. A CINC-1/CXCL1 concentration in BALF. B CINC-2/CXCL3 concentration in BALF. C CXCL5 concentration in BALF. D HO-1 concentration in lung tissue. The expressions of CINC-1/CXCL1, CINC-2/CXCL3 and CXCL5 in BALF in the exposed groups were persistently higher compared to the control group in a dose dependent-manner during 1 month after the exposure. Data are presented as mean ± SE (* p < 0.05 and ** p < 0.01 indicate that the values are significantly higher than control group. † p < 0.05 and †† p < 0.01 indicate that the values are significantly lower than control group.)

Article Snippet: Immunostaining for CXCL5 was performed with rabbit anti-mouse CXCL5 polyclonal antibody (1:200 dilution, bs-2549R; Bioss Inc., Woburn, MA, USA), while using the lung tissue samples from the 1.0 mg CL-PAA-exposure group of one month after intratracheal instillation.

Techniques: Concentration Assay

Journal: Particle and Fibre Toxicology

Article Title: Inflammogenic effect of polyacrylic acid in rat lung following intratracheal instillation

doi: 10.1186/s12989-022-00448-z

Figure Lengend Snippet: Description of chemokine genes related to ‘inflammatory response’ among 58 genes upregulated ≧ eightfold

Article Snippet: Immunostaining for CXCL5 was performed with rabbit anti-mouse CXCL5 polyclonal antibody (1:200 dilution, bs-2549R; Bioss Inc., Woburn, MA, USA), while using the lung tissue samples from the 1.0 mg CL-PAA-exposure group of one month after intratracheal instillation.

Techniques:

Journal: Particle and Fibre Toxicology

Article Title: Inflammogenic effect of polyacrylic acid in rat lung following intratracheal instillation

doi: 10.1186/s12989-022-00448-z

Figure Lengend Snippet: Representative images of CXCL5 immunostaining in lung tissue at 1 month after exposure to CL-PAA. A control lung (HE staining), B 1.0 mg CL-PAA-exposed lung (HE staining), C control lung (CXCL5 immunostaining), D 1.0 mg CL-PAA-exposed lung (CXCL5 immunostaining). Positive cells of CXCL5 immunostaining on 1.0 mg CL-PAA-exposed lung were mainly macrophages (blue arrow heads) (internal scale bar = 250 μm for all)

Article Snippet: Immunostaining for CXCL5 was performed with rabbit anti-mouse CXCL5 polyclonal antibody (1:200 dilution, bs-2549R; Bioss Inc., Woburn, MA, USA), while using the lung tissue samples from the 1.0 mg CL-PAA-exposure group of one month after intratracheal instillation.

Techniques: Immunostaining, Staining

Journal: The Journal of Clinical Investigation

Article Title: A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy

doi: 10.1172/JCI133055

Figure Lengend Snippet: (A) Schematic overview of the adaptive resistance pathway. (B) RNA-Seq differential gene expression analysis of tumor tissues following treatment of the autochthonous BRAFV600E PTEN–/– melanoma model with anti–PD-1 Ab therapy versus IgG isotype control (Ctrl) (n = 3). (C) qRT-PCR analysis of target genes of interest in serial tumor fine-needle aspiration (FNA) biopsy specimens harvested from the transgenic BRAFV600E PTEN–/– melanoma model treated with anti–PD-1 Ab versus IgG isotype control (n = 5). (D) Gr-1 immunohistochemical analysis of transgenic BRAFV600E PTEN–/– melanoma tissues following treatment with anti–PD-1 Ab versus IgG isotype control. Original magnification, ×40. Gr-1 staining is shown in red. Images are representative of 3 tumors per group. (E) PMN-MDSC flow cytometric analysis of transgenic BRAFV600E PTEN–/– melanoma tissues following treatment with anti–PD-1 Ab versus IgG isotype control. PMN-MDSCs were defined as live+CD45+CD11b+Ly6G+Ly6CintF4/80– cells. Shown are a representative flow dot plot and quantification graph of PMN-MDSC flow cytometric data (n = 5). (F) qRT-PCR analysis of CXCR2 ligands in BRAFV600E PTEN–/– melanoma tissues treated with anti–PD-1 Ab following CD8+ T cell ablation in vivo (n = 3). (G) In vivo tumor study of BRAFV600E PTEN–/– melanoma genetically silenced for CXCL5. Quantitation of tumor-infiltrating PMN-MDSCs by flow cytometry is shown along with an in vivo tumor growth curve of CXCL5-silenced BRAFV600E PTEN–/– melanoma versus BRAFV600E PTEN–/– NTC melanoma control tumors treated with anti–PD-1 Ab. Data were normalized to tumors treated with IgG isotype control (n = 5). (H) Combination treatment with anti–PD-1 Ab and CXCR2 inhibitor (CXCR2i) in an in vivo BRAFV600E PTEN–/– melanoma study (n = 5). Graphs show flow cytometric analysis of tumor-infiltrating PMN-MDSCs and live+CD45+CD3+CD8+ T cells. *P < 0.05, **P < 0.005, and ***P < 0.0005, by Student’s t test with Holm-Sidak post hoc correction for multiple comparisons (B, C, and F), Student’s t test (E and G), or 1-way ANOVA with Sidak’s post hoc multiple comparisons test (H). See also Supplemental Figures 1, 2, and 5C.

Article Snippet: The following Abs were used: anti–β-actin, mouse mAb (Santa Cruz Biotechnology, sc-47778); anti-NLRP3, rabbit mAb (Cell Signaling Technology, 15101S); anti-ASC, mouse mAb (Santa Cruz Biotechnology, sc-514414); anti–caspase-1 p20, mouse mAb (Adipogen, AG-20B-0042-C100); anti–caspase-3, rabbit polyclonal Ab (Cell Signaling Technology, 9662S); anti-HSP70, mouse mAb (Santa Cruz Biotechnology, sc-66048); anti-CXCL5, goat polyclonal Ab (R&D Systems, AF433); anti-CXCL5, rabbit polyclonal Ab (LSBio, LS-c293780); anti–YAP/TAZ, rabbit mAb (Cell Signaling Technology, 8418S); anti-Wnt5a, mouse mAb (Santa Cruz Biotechnology, sc-365370); anti-GAPDH, mouse mAb (Santa Cruz Biotechnology, sc-32233); CD8a, rabbit mAb (Cell Signaling Technology, 989415); InVivoMAb anti–human PD-L1 Ab (Bio X Cell, BE0285); InVivoMAb anti–mouse PD-L1 Ab (Bio X Cell, BE0101); InVivoMAb anti–mouse PD-1 Ab (Bio X Cell, BE0146); InVivoMAb rat IgG2a isotype control Ab, clone: 2A3 (Bio X Cell, BE0089); anti–mouse CD8 Ab from hybridoma, (Duke Cell Culture Facility, clone 53.6.7); anti-OVA Ab (Santa Cruz Biotechnology, sc-65984); anti–IL-1β, mouse mAb (Cell Signaling Technology, 12242); anti–LY6G–Gr1 Ab (Abcam, ab25377); goat anti–rat IgG H&L Ab (MilliporeSigma, AP136P).

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Immunohistochemical staining, Staining, In Vivo, Quantitation Assay, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy

doi: 10.1172/JCI133055

Figure Lengend Snippet: (A) TCGA human melanoma database gene expression analysis of CXCL5, CXCL2, and CXCR2 association with WNT5A. (B) Whole tumor tissue Western blot analysis of Wnt5a, YAP1, CXCL5, and vinculin and β-actin (used as loading controls). Blot is representative of 3 independent experiments. (C) Plasma CXCL5 ELISA following anti–PD-1 Ab therapy versus IgG isotype control therapy in the transgenic BRAFV600E PTEN–/– melanoma model (n = 6). Data are representative of 3 independent experiments. (D) qRT-PCR analysis of Cxcl1, Cxcl2, and Cxcl5 in the BRAFV600E PTEN–/– melanoma cell line following treatment with rWnt5a versus vehicle control (n = 3). (E) Western blot analysis of YAP1 expression in total cellular lysates (top) and nuclear lysates (middle) following treatment of BRAFV600E PTEN–/– melanoma cells with rWnt5a at various time points. Bottom blot shows Wnt5a induction of CXCL5 with or without verteporfin (YAP inhibitor) or XAV939 (β-catenin inhibitor). Blots shown are representative of 3 independent experiments. UT, untreated or vehicle control. (F) qRT-PCR analysis of Cxcl5 in BRAFV600E PTEN–/– NTC and Wnt5a-silenced BRAFV600E PTEN–/– melanoma cells (BRAFV600E PTEN–/– Wnt5aKD). Blot shows secreted CXCL5 in BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– Wnt5aKD cells (n = 3). (G) IHC for CXCL5 (red) in BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– Wnt5aKD tumor cells. Images are representative of 3 tumors. White arrows indicate CXCL5+ tumor cells. Original magnification, ×20. (H) IHC for Gr-1 in BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– Wnt5aKD tumor cells. Original magnification, ×20. Plots show PMN-MDSC flow cytometric analysis of BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– Wnt5aKD tumors (n = 3). (I) PMN-MDSC flow cytometric analysis of BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– Wnt5aKD tumors following treatment with anti–PD-1 Ab versus IgG isotype control (n = 5). (J) Tumor volume change based on anti–PD-1 Ab/IgG control ratios for BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– Wnt5aKD tumors (n = 5). α, anti. UT, untreated control. Kendall’s tau correlation coefficient was calculated for A. *P < 0.05 and ***P < 0.0005, by Student’s t test (C, D, and I) and 1-way ANOVA with Sidak’s post hoc multiple comparisons test (F). See also Supplemental Figure 3.

Article Snippet: The following Abs were used: anti–β-actin, mouse mAb (Santa Cruz Biotechnology, sc-47778); anti-NLRP3, rabbit mAb (Cell Signaling Technology, 15101S); anti-ASC, mouse mAb (Santa Cruz Biotechnology, sc-514414); anti–caspase-1 p20, mouse mAb (Adipogen, AG-20B-0042-C100); anti–caspase-3, rabbit polyclonal Ab (Cell Signaling Technology, 9662S); anti-HSP70, mouse mAb (Santa Cruz Biotechnology, sc-66048); anti-CXCL5, goat polyclonal Ab (R&D Systems, AF433); anti-CXCL5, rabbit polyclonal Ab (LSBio, LS-c293780); anti–YAP/TAZ, rabbit mAb (Cell Signaling Technology, 8418S); anti-Wnt5a, mouse mAb (Santa Cruz Biotechnology, sc-365370); anti-GAPDH, mouse mAb (Santa Cruz Biotechnology, sc-32233); CD8a, rabbit mAb (Cell Signaling Technology, 989415); InVivoMAb anti–human PD-L1 Ab (Bio X Cell, BE0285); InVivoMAb anti–mouse PD-L1 Ab (Bio X Cell, BE0101); InVivoMAb anti–mouse PD-1 Ab (Bio X Cell, BE0146); InVivoMAb rat IgG2a isotype control Ab, clone: 2A3 (Bio X Cell, BE0089); anti–mouse CD8 Ab from hybridoma, (Duke Cell Culture Facility, clone 53.6.7); anti-OVA Ab (Santa Cruz Biotechnology, sc-65984); anti–IL-1β, mouse mAb (Cell Signaling Technology, 12242); anti–LY6G–Gr1 Ab (Abcam, ab25377); goat anti–rat IgG H&L Ab (MilliporeSigma, AP136P).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy

doi: 10.1172/JCI133055

Figure Lengend Snippet: (A) RNA-Seq GSEA showing top 12 pathways enriched in autochthonous BRAFV600E PTEN–/– melanomas following escape from anti–PD-1 Ab therapy. Arrows indicate pathways associated with cellular stress (n = 3/group). (B) SILAC-AHA LC-MS/MS secretome analysis of resected autochthonous BRAFV600E PTEN–/– melanoma tissues following anti–PD-1 Ab therapy versus IgG isotype control. Secreted protein levels were normalized to the number of cells (n = 3/group). (C) Plasma HSP70 ELISA analysis following anti–PD-1 versus IgG isotype control treatment of autochthonous BRAFV600E PTEN–/– melanoma-bearing mice (n = 6). (D) qRT-PCR analysis of TLR expression in BRAFV600E PTEN–/– melanoma cells. Data were normalized to Tlr9 expression levels (n = 3). (E) Treatment of BRAFV600E PTEN–/– melanoma cells with titrated concentrations of recombinant HSP70 (rHSP70) followed by Wnt5a Western blot analysis of total cell lysates and supernatant (SNT). Blots are representative of 2 independent experiments. (F) Treatment of BRAFV600E PTEN–/– melanoma cells with titrated concentrations of the HSP70 inhibitor VER155008 (HSP70i). Blots are representative of 2 independent experiments. (G) Treatment of BRAFV600E PTEN–/– NTC cells with rHSP70 with or without the TLR4 inhibitor CLI-095 (TLR4i) and treatment of Tlr4-silenced BRAFV600E PTEN–/– melanoma cells (TLR4KD) with HSP70 followed by Western blotting for Wnt5a. Blots are representative of 3 independent experiments. (H) BRAFV600E PTEN–/– melanoma growth curve following treatment with TLR4 siRNA versus control siRNA (n = 5). (I) Whole-tissue Western blot analysis of Wnt5a, CXCL5, and β-actin in TLR4 siRNA–treated and control siRNA–treated BRAFV600E PTEN–/– melanomas. Data are representative of 2 independent experiments. (J) Top: PMN-MDSC flow cytometric analysis of TLR4 siRNA– and control siRNA–treated BRAFV600E PTEN–/– melanomas (n = 4). Bottom: CD8+ T cell flow cytometric analysis of TLR4 siRNA– and control siRNA–treated BRAFV600E PTEN–/– melanomas (n = 4). *P < 0.05, by Student’s t test for comparison of treatment groups. See also Supplemental Figure 4.

Article Snippet: The following Abs were used: anti–β-actin, mouse mAb (Santa Cruz Biotechnology, sc-47778); anti-NLRP3, rabbit mAb (Cell Signaling Technology, 15101S); anti-ASC, mouse mAb (Santa Cruz Biotechnology, sc-514414); anti–caspase-1 p20, mouse mAb (Adipogen, AG-20B-0042-C100); anti–caspase-3, rabbit polyclonal Ab (Cell Signaling Technology, 9662S); anti-HSP70, mouse mAb (Santa Cruz Biotechnology, sc-66048); anti-CXCL5, goat polyclonal Ab (R&D Systems, AF433); anti-CXCL5, rabbit polyclonal Ab (LSBio, LS-c293780); anti–YAP/TAZ, rabbit mAb (Cell Signaling Technology, 8418S); anti-Wnt5a, mouse mAb (Santa Cruz Biotechnology, sc-365370); anti-GAPDH, mouse mAb (Santa Cruz Biotechnology, sc-32233); CD8a, rabbit mAb (Cell Signaling Technology, 989415); InVivoMAb anti–human PD-L1 Ab (Bio X Cell, BE0285); InVivoMAb anti–mouse PD-L1 Ab (Bio X Cell, BE0101); InVivoMAb anti–mouse PD-1 Ab (Bio X Cell, BE0146); InVivoMAb rat IgG2a isotype control Ab, clone: 2A3 (Bio X Cell, BE0089); anti–mouse CD8 Ab from hybridoma, (Duke Cell Culture Facility, clone 53.6.7); anti-OVA Ab (Santa Cruz Biotechnology, sc-65984); anti–IL-1β, mouse mAb (Cell Signaling Technology, 12242); anti–LY6G–Gr1 Ab (Abcam, ab25377); goat anti–rat IgG H&L Ab (MilliporeSigma, AP136P).

Techniques: RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Recombinant, Western Blot, Comparison

Journal: The Journal of Clinical Investigation

Article Title: A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy

doi: 10.1172/JCI133055

Figure Lengend Snippet: (A) Plasma HSP70 ELISA analysis following the growth of BRAFV600E PTEN–/– NTC or Nlrp3-silenced BRAFV600E PTEN–/– melanomas (n = 5). (B) qRT-PCR analysis of CXCR2-dependent chemokine expression in BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– NLRP3KD melanomas (n = 3). (C) Flow cytometric analysis of CD8+ T cells in resected BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– NLRP3KD melanomas (n = 5). Flow cytometric analysis of PMN-MDSCs in resected BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– NLRP3KD melanomas (n = 5). (D) Tumor growth curve of BRAFV600E PTEN–/– NTC and BRAFV600E PTEN–/– NLRP3KD melanomas (n = 5). (E) Treatment of syngeneic BRAFV600E PTEN–/– melanomas with IgG isotype control Ab (200 μg i.p. every 3 days), NLRP3 inhibitor (10 μg MCC950 i.p. every 3 days), anti–PD-1 Ab (200 μg i.p. every 3 days), or NLRP3 inhibitor and anti–PD-1 Ab combination therapy (n = 8). (F) Representative flow cytometric dot plots of PMN-MDSCs and CD8+ T cells in resected BRAFV600E PTEN–/– melanomas following treatment with IgG isotype control Ab, NLRP3 inhibitor, anti–PD-1 Ab, or NLRP3 inhibitor and anti–PD-1 Ab combination therapy. Graphs show flow cytometric analysis of tumor-infiltrating PMN-MDSCs and CD44+CD8+ T cells. (G) Whole tumor tissue Western blot analysis for pro–caspase-1, caspase-1 p20, and Wnt5a following in vivo treatment with IgG isotype control, anti–PD-1 Ab, or combined anti–PD-1 Ab and NLRP3 inhibitor. Blots are representative of 2 independent experiments. (H) qRT-PCR analysis of Cxcl5 and granzyme B (Gzmb) expression in resected BRAFV600E PTEN–/– melanoma tissues (n = 5). *P < 0.05, **P < 0.005, and ***P < 0.0005, by Student’s t test (A–D) and 1-way ANOVA with Sidak’s post hoc multiple comparisons test (E, F, and H). See also Supplemental Figure 7.

Article Snippet: The following Abs were used: anti–β-actin, mouse mAb (Santa Cruz Biotechnology, sc-47778); anti-NLRP3, rabbit mAb (Cell Signaling Technology, 15101S); anti-ASC, mouse mAb (Santa Cruz Biotechnology, sc-514414); anti–caspase-1 p20, mouse mAb (Adipogen, AG-20B-0042-C100); anti–caspase-3, rabbit polyclonal Ab (Cell Signaling Technology, 9662S); anti-HSP70, mouse mAb (Santa Cruz Biotechnology, sc-66048); anti-CXCL5, goat polyclonal Ab (R&D Systems, AF433); anti-CXCL5, rabbit polyclonal Ab (LSBio, LS-c293780); anti–YAP/TAZ, rabbit mAb (Cell Signaling Technology, 8418S); anti-Wnt5a, mouse mAb (Santa Cruz Biotechnology, sc-365370); anti-GAPDH, mouse mAb (Santa Cruz Biotechnology, sc-32233); CD8a, rabbit mAb (Cell Signaling Technology, 989415); InVivoMAb anti–human PD-L1 Ab (Bio X Cell, BE0285); InVivoMAb anti–mouse PD-L1 Ab (Bio X Cell, BE0101); InVivoMAb anti–mouse PD-1 Ab (Bio X Cell, BE0146); InVivoMAb rat IgG2a isotype control Ab, clone: 2A3 (Bio X Cell, BE0089); anti–mouse CD8 Ab from hybridoma, (Duke Cell Culture Facility, clone 53.6.7); anti-OVA Ab (Santa Cruz Biotechnology, sc-65984); anti–IL-1β, mouse mAb (Cell Signaling Technology, 12242); anti–LY6G–Gr1 Ab (Abcam, ab25377); goat anti–rat IgG H&L Ab (MilliporeSigma, AP136P).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot, In Vivo