Human PD-1 Antibody

Detection of Human PD‑1 by Western Blot. Western blot shows lysates of human peripheral blood mononuclear cells (PBMC) untreated (-) or treated (+) with 1 µg/mL PHA for 5 days. PVDF Membrane was probed with 1 µg/mL of Human PD-1 Monoclonal Antibody (Catalog # MAB1086) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for PD-1 at approximately 33 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human PD-1 by Immunohistochemistry The role of IL‐6 signaling in the upregulation of PD‐L1 and downregulation of NKG2D ligands in CRPC cells. (A) PD‐L1 level in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cell lines (left panel, mRNA level; right panel, protein level). (B) PD‐L1 IHC staining of tumor tissues. Error bars and significance values were obtained by counting positively stained cells in one randomly chosen phase of slides of three different stains. Magnification, 20× (inlet, 100×). (C) Blocking of IL‐6 Ab by neutralizing Ab of IL‐6 and the effect on PD‐L1 level in C4‐2sc and CWRsc cells. Cells were treated with either IL‐6 Ab or control IgG, total RNA extracted, cDNA converted, and the expression of PD‐L1 was compared in qPCR analyses. (D) PD‐L1 level in parental C4‐2 and CWR22Rv1 cells upon the addition of rhIL‐6. Parental cells (C4‐2P and CWR22Rv1P) were treated with rhIL‐6 (20 ng·mL−1) and PD‐L1 mRNA level was analyzed. (E) IHC staining of CRPC patient tumor samples. Two sets of adjacent tumor tissues (both samples, CRPC stage, Gleason score 8, patient age 70, Ningbo hospital in China) were stained with IL‐6 and PD‐L1. Arrows indicate the area showing positive staining of two molecules. (F) NKG2D ligand levels in IL‐6‐expressing cells and in IL‐6‐knockdown cells. Levels of five NKG2D ligands in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cells were analyzed in qPCR analyses. (G) NKG2D ligand levels in parental C4‐2 and CWR22Rv1 cells upon the addition of rhIL‐6. Parental cells (C4‐2P and CWR22Rv1P) were treated with rhIL‐6 (20 ng·mL−1) and the NKG2D ligand levels (mRNA) were analyzed. (H) Flow cytometric analyses of NKG2D and PD‐1 on NK cells. Left two panels, primary NK cells were stained with PE‐NKG2D or APC‐PD‐1 and positive staining was analyzed. Right two panels, flow cytometric analyses of PD‐1 on NK cells, after coculture with tumor cells (6 h of incubation). Primary NK cells were added into tumor cells (1 : 1 ratio, tumor cells/NK cells) and collected after 6 h of incubation. PD‐1 levels in the collected NK cells were analyzed in flow cytometric analysis (using APC‐PD‐1 Ab). *P < 0.05, **P < 0.01, ***P < 0.001. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28865178), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human PD-1 by Western Blot PD-L1/NKG2D ligand levels in radioresistant NSCLC sub-line cells and radioresistant cell-derived tumors (compared to parental cells and parental cell-derived tumors)(A) An illustration of radioresistant sub-line development procedure. (B) Clonogenic assay showing radioresistance of A549R26-1 and H157R24-1 sublines vs. respective parental cells. 100 to 1,000 cells (A549P/A549R26-1 and H157P/H157R24-1) were plated and cell survivals after different doses of radiation treatment were analyzed in clonogenic assay. (C) gamma H2AX IF staining. The numbers of gamma H2AX foci (A549P/A549R26-1 and H157P/H157R24-1) at different time points after radiation (6 Gy) were analyzed. (D) PD-L1 expression in parental and radioresistant cells (upper panel, qPCR data; lower panel, Western blot data). (E) Flow cytometry results analyzing surface PD-L1 levels in parental (red) and radioresistant (blue) cells. (F) Expression of NKG2D ligands (mRNA) in parental vs. radioresistant cells. (G) Western blot analysis results showing the PD-L1 levels in injected luc-549R26-1 and luc-A549P cells. (H) Mice studies testing radioresistance. Tumor regression differences at each time point in A549P-xenografts vs. A549R26-1 xenografts, with or without radiation treatments (5 Gy × 5 days) is shown. Y-axis represents fold of tumor growth based on luminescence measurement. (I) IHC staining of PD-L1 in tumor tissues obtained from A549P cells-derived and A549R26-1 cells-derived xenografts. *p < 0.05, **p < 0.01, ***p < 0.001 Image collected and cropped by CiteAb from the following publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.19193), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human PD-1 by Western Blot PD-L1/NKG2D ligand levels in radioresistant NSCLC sub-line cells and radioresistant cell-derived tumors (compared to parental cells and parental cell-derived tumors)(A) An illustration of radioresistant sub-line development procedure. (B) Clonogenic assay showing radioresistance of A549R26-1 and H157R24-1 sublines vs. respective parental cells. 100 to 1,000 cells (A549P/A549R26-1 and H157P/H157R24-1) were plated and cell survivals after different doses of radiation treatment were analyzed in clonogenic assay. (C) gamma H2AX IF staining. The numbers of gamma H2AX foci (A549P/A549R26-1 and H157P/H157R24-1) at different time points after radiation (6 Gy) were analyzed. (D) PD-L1 expression in parental and radioresistant cells (upper panel, qPCR data; lower panel, Western blot data). (E) Flow cytometry results analyzing surface PD-L1 levels in parental (red) and radioresistant (blue) cells. (F) Expression of NKG2D ligands (mRNA) in parental vs. radioresistant cells. (G) Western blot analysis results showing the PD-L1 levels in injected luc-549R26-1 and luc-A549P cells. (H) Mice studies testing radioresistance. Tumor regression differences at each time point in A549P-xenografts vs. A549R26-1 xenografts, with or without radiation treatments (5 Gy × 5 days) is shown. Y-axis represents fold of tumor growth based on luminescence measurement. (I) IHC staining of PD-L1 in tumor tissues obtained from A549P cells-derived and A549R26-1 cells-derived xenografts. *p < 0.05, **p < 0.01, ***p < 0.001 Image collected and cropped by CiteAb from the following publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.19193), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human PD-1 by Western Blot Revealing signaling pathways that are responsible for the PD-L1 level increase/NKG2D ligand level decrease in A549R26-1 and H157R24-1 cells(A) Western blot analyses showing expression/activation of several signaling molecules in parental and radioresistant cells. (B) Western blot analyses showing inhibition of each pathway in A549R26-1 cells upon inhibitor treatment. (C and D) qPCR analyses (C) and Western blot analyses (D) of PD-L1 levels in A549R26-1 and H157R24-1 cells upon treatment with inhibitors of indicated signaling pathways. (E) Western blot analysis showing p-MEK/p-Erk levels after adding IL-6 Ab (control IgG as control) in radioresistant cells. (F) A figure describing the mechanism leading to the resistance to NK cell cytotoxicity. *p < 0.05, **p < 0.01, ***p < 0.001. Image collected and cropped by CiteAb from the following publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.19193), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human PD-1 by Western Blot Revealing signaling pathways that are responsible for the PD-L1 level increase/NKG2D ligand level decrease in A549R26-1 and H157R24-1 cells(A) Western blot analyses showing expression/activation of several signaling molecules in parental and radioresistant cells. (B) Western blot analyses showing inhibition of each pathway in A549R26-1 cells upon inhibitor treatment. (C and D) qPCR analyses (C) and Western blot analyses (D) of PD-L1 levels in A549R26-1 and H157R24-1 cells upon treatment with inhibitors of indicated signaling pathways. (E) Western blot analysis showing p-MEK/p-Erk levels after adding IL-6 Ab (control IgG as control) in radioresistant cells. (F) A figure describing the mechanism leading to the resistance to NK cell cytotoxicity. *p < 0.05, **p < 0.01, ***p < 0.001. Image collected and cropped by CiteAb from the following publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.19193), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human PD-1 by Western Blot The role of IL‐6 signaling in the upregulation of PD‐L1 and downregulation of NKG2D ligands in CRPC cells. (A) PD‐L1 level in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cell lines (left panel, mRNA level; right panel, protein level). (B) PD‐L1 IHC staining of tumor tissues. Error bars and significance values were obtained by counting positively stained cells in one randomly chosen phase of slides of three different stains. Magnification, 20× (inlet, 100×). (C) Blocking of IL‐6 Ab by neutralizing Ab of IL‐6 and the effect on PD‐L1 level in C4‐2sc and CWRsc cells. Cells were treated with either IL‐6 Ab or control IgG, total RNA extracted, cDNA converted, and the expression of PD‐L1 was compared in qPCR analyses. (D) PD‐L1 level in parental C4‐2 and CWR22Rv1 cells upon the addition of rhIL‐6. Parental cells (C4‐2P and CWR22Rv1P) were treated with rhIL‐6 (20 ng·mL−1) and PD‐L1 mRNA level was analyzed. (E) IHC staining of CRPC patient tumor samples. Two sets of adjacent tumor tissues (both samples, CRPC stage, Gleason score 8, patient age 70, Ningbo hospital in China) were stained with IL‐6 and PD‐L1. Arrows indicate the area showing positive staining of two molecules. (F) NKG2D ligand levels in IL‐6‐expressing cells and in IL‐6‐knockdown cells. Levels of five NKG2D ligands in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cells were analyzed in qPCR analyses. (G) NKG2D ligand levels in parental C4‐2 and CWR22Rv1 cells upon the addition of rhIL‐6. Parental cells (C4‐2P and CWR22Rv1P) were treated with rhIL‐6 (20 ng·mL−1) and the NKG2D ligand levels (mRNA) were analyzed. (H) Flow cytometric analyses of NKG2D and PD‐1 on NK cells. Left two panels, primary NK cells were stained with PE‐NKG2D or APC‐PD‐1 and positive staining was analyzed. Right two panels, flow cytometric analyses of PD‐1 on NK cells, after coculture with tumor cells (6 h of incubation). Primary NK cells were added into tumor cells (1 : 1 ratio, tumor cells/NK cells) and collected after 6 h of incubation. PD‐1 levels in the collected NK cells were analyzed in flow cytometric analysis (using APC‐PD‐1 Ab). *P < 0.05, **P < 0.01, ***P < 0.001. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28865178), licensed under a CC-BY license. Not internally tested by R&D Systems.