Immunohistochemistry:Article Title: Targeting the STAT5A/IDO1 Axis Overcomes Radiotherapy Resistance and Reverses Immunosuppressive Tumor Microenvironment in Non-Small Cell Lung Cancer
Article Snippet: Background: Radiotherapy (RT) combined with immunotherapy is an important point of contention in non-small cell lung cancer (NSCLC) research.. As an important metabolic mediator of antitumor immunity, indoleamine-2,3-dioxygenase 1 (IDO1) is reportedly upregulated in a variety of cancers.. However, the regulatory mechanism and clinical Manuscript Click here to view linked References This preprint research paper has not been peer reviewed.
Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO 2 . .. The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), anti-PD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm-30107M, Bioss antibodies, Beijing, China), rabbit anti-RGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PD-L2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade.
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO2. .. Antibodies and reagents The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), antiPD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm30107M, Bioss antibodies, Beijing, China), rabbit antiRGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PDL2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/ Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/ Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Western Blot:Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO 2 . .. The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), anti-PD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm-30107M, Bioss antibodies, Beijing, China), rabbit anti-RGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PD-L2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade.
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO2. .. Antibodies and reagents The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), antiPD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm30107M, Bioss antibodies, Beijing, China), rabbit antiRGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PDL2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/ Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/ Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Immunofluorescence:Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO 2 . .. The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), anti-PD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm-30107M, Bioss antibodies, Beijing, China), rabbit anti-RGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PD-L2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade.
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO2. .. Antibodies and reagents The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), antiPD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm30107M, Bioss antibodies, Beijing, China), rabbit antiRGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PDL2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/ Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/ Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Chromatin Immunoprecipitation:Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO 2 . .. The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), anti-PD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm-30107M, Bioss antibodies, Beijing, China), rabbit anti-RGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PD-L2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade.
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO2. .. Antibodies and reagents The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), antiPD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm30107M, Bioss antibodies, Beijing, China), rabbit antiRGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PDL2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/ Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/ Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Flow Cytometry:Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO 2 . .. The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), anti-PD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm-30107M, Bioss antibodies, Beijing, China), rabbit anti-RGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PD-L2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
Article Title: PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade.
Article Snippet: These cells were cultured in Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO2. .. Antibodies and reagents The antibodies used in this study were as follows: rabbit anti-human PD-L2 (#82723, Cell Signaling Technology, Beverly, MA, USA; 1:100 for immunohistochemistry (IHC)), rabbit anti-human PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), rabbit anti-human FOXP3 (#98377, Cell Signaling Technology, Beverly, MA, USA; 1:100 for IHC), anti-p-p65 (#3033, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot), antiPD-L2 (#ab87662, Abcam, Cambridge, MA, USA; 1:1000 for western blot), mouse anti-PD-L2 (#bsm30107M, Bioss antibodies, Beijing, China), rabbit antiRGMB (#bs-11474R, Bioss antibodies, Beijing, China; 1:1000 for western blot), anti-RGMB (#A12859, Abclonal, China; 1:1000 for western blot), anti-PDL2 (#ab288298, Abcam, Cambridge, MA, USA; 1:50 for Co-immunoprecipitation); anti-β-Actin (#A1978, Sigma, St. Louis, MO, USA; 1:5000 for western blot), anti-PD-L2 (#sc-57398, Santa cruz, USA; 1:50 for IHC), anti-PD-L2 (#NBP1-76770, Novus iologicals, LLC, USA; 1:100 for immunofluorescence assay), anti-p65 (#A19653, Abclonal, China; 1:1000 for western blot), anti-AhR (#83200, Cell Signaling Technology, Beverly, MA, USA; 1:1000 for western blot, 1:50 for Chromatin immunoprecipitation (CHIP)), Zombie AquaTM dye (#423101,Biolegend; 1:1000 for flow cytometry), BUV395 anti-mouse CD45 (#745698, BD OptiBuild, 1:100 for flow cytometry), APC anti-mouse CD4 (#100411,Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD8a (#100721,Biolegend; 1:20 for flow cytometry), APC/Cy7 anti-mouse CD11b (#101225, Biolegend; 1:20 for flow cytometry), PE/ Cy5 anti-mouse F4/80 (#123111, Biolegend; 1:20 for flow cytometry), PE anti-mouse Gr1 (#108407, Biolegend; 1:20 for flow cytometry), APC anti-mouse CD206 (#141707, Biolegend; 1:20 for flow cytometry), PE/ Cy7 anti-mouse MHCII (#107629, Biolegend; 1:20 for flow cytometry), PE/Cy7 anti-mouse CD86 (#105013, Biolegend; 1:20 for flow cytometry), PE anti-mouse FOXP3 (#12-4776-41, eBioscience; 1:20 for flow cytometry), PE anti-human PD-L1 (#329705, Biolegend; 1:20 for flow cytometry), and PE anti-human PD-L2 (#329605, Biolegend; 1:20 for flow cytometry). .. Benzo(a)pyrene (#B1760), Alpha-Naphthoflavone (ANF; #N5757), Puromycin, and RNase A were purchased from Sigma-Aldrich.
other:Article Title: Factor V is an immune inhibitor that is expressed at increased levels in leukocytes of patients with severe Covid-19
Article Snippet: Rabbit anti-human FoxP3 (Cell signalling technology,Hitchin, UK).
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![(A) Representative flow cytometry plot and gating strategy for determining ST2 + cell identity in day 2 oral wounds. (B) EPCAM + (epithelial), PDGFRA + (fibroblast), CD31 + (endothelial), and CD45 + (leukocyte) cells that express ST2, normalized by total ST2 + cell count per time point. (C) scRNA-seq analysis and UMAP plot of CD45 + sorted cells from day 2 wounds of control Il33 f/f mice (Ctrl) and experimental ΔIL33 Col1α2 mice. 8–10 wounds from 4–5 mice were pooled per condition. (D) Left: UMAP of subset clusters that express Il1rl1 + (encoding ST2). Right: feature plot of Il1rl1 expression in three clusters. (E) Dot plot for genes that are enriched in Treg cells, including <t>Foxp3</t> . (F) Il1rl1 + cell numbers in each cluster normalized by total number of Il1rl1 + cells. Treg, regulatory T cell; MC, mast cell; ILC2, type 2 innate lymphoid cell. (G) Pseudobulk analysis of DEGs, comparing Treg cells from control versus ΔIL33 Col1α2 groups. (H) GO analysis for the upregulated gene list in control Treg cells. Biological process terms associated with immune responses are shown. (I) CellChat analysis of ligand-receptor interaction pairs. Treg cells are set as the ligand source and other leukocytes as the recipients. Communication probability from the control and ΔIL33 Col1α2 (conditional knockout [cKO]) groups are shown. (J) Violin plots of Mif and Tgfb1 expression in Treg cell cluster from control and ΔIL33 Col1α2 groups. (K) Flow cytometry plots of MIF and TGF-β1 expression in FOXP3 + Treg cells from day 2 wounds of control or ΔIL33 Col1α2 mice. (L) Mean fluorescent intensity (MFI) for MIF or TGF-β1 expression in FOXP3 + Treg cells in day 2 wounds. Data represent mean ± SEM; n = 3–5 mice per group. Student’s t test, * p < 0.05.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5373/pmc12955373/pmc12955373__nihms-2142960-f0004.jpg)
