PD-1 Antibody (192106) [Unconjugated]

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.

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 (//pubmed.ncbi.nlm.nih.gov/28865178), licensed under a CC-BY license. Not internally tested by R&D Systems.

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 (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.19193), licensed under a CC-BY license. Not internally tested by R&D Systems.

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 (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.19193), licensed under a CC-BY license. Not internally tested by R&D Systems.

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 (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.19193), licensed under a CC-BY license. Not internally tested by R&D Systems.

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 (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.19193), licensed under a CC-BY license. Not internally tested by R&D Systems.

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 (//pubmed.ncbi.nlm.nih.gov/28865178), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB
• Clone: 192106
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: LYOPH

PD-1 Antibody (192106) [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant human PD-1
  Leu25-Gln167
  Accession # Q8IX89
• Specificity: Detects human PD-1 in direct ELISAs and Western blots. In direct ELISAs and Western blots, no cross-reactivity with recombinant human (rh) CD28, rhCTLA4, rhICOS, or recombinant mouse PD-1 is observed.
• Source: N/A
• Isotype: IgG2b
• Clonality: Monoclonal
• Host: Mouse
• Gene: PDCD1
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Western Blot 1 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for PD-1 Antibody (192106) [Unconjugated]

• CD279 antigen
• CD279
• hPD-1
• PD1
• PD-1
• PD1hPD-l
• PDCD1
• programmed cell death 1
• programmed cell death protein 1
• Protein PD-1
• SLEB2

Background

Programmed Death-1 (PD-1) is a type I transmembrane protein belonging to the CD28/CTLA-4 family of immunoreceptors that mediate signals for regulating immune responses (1). Members of the CD28/CTLA-4 family have been shown to either promote T cell activation (CD28 and ICOS) or downregulate T cell activation (CTLA-4 and PD-1) (2). PD-1 is expressed on activated T cells, B cells, myeloid cells, and on a subset of thymocytes. In vitro, ligation of PD-1 inhibits TCR-mediated T cell proliferation and production of IL-1, IL-4, IL-10, and IFN-gamma . In addition, PD-1 ligation also inhibits BCR mediated signaling. PD-1 deficient mice have a defect in peripheral tolerance and spontaneously develop autoimmune diseases (2, 3). Two B7 family proteins, PD-L1 (also called B7-H1) and PD-L2 (also known as B7-DC), have been identified as PD-1 ligands. Unlike other B7 family proteins, both PD‑L1 and PD‑L2 are expressed in a wide variety of normal tissues including heart, placenta, and activated spleens (4). The wide expression of PD-L1 and PD-L2 and the inhibitor effects on PD-1 ligation indicate that PD-1 might be involved in the regulation of peripheral tolerance and may help prevent autoimmune diseases (2). The human PD-1 gene encodes a 288 amino acid (aa) protein with a putative 20 aa signal peptide, a 148 aa extracellular region with one immunoglobulin-like V‑type domain, a 24 aa transmembrane domain, and a 95 aa cytoplasmic region. The cytoplasmic tail contains two tyrosine residues that form the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) that are important in mediating PD-1 signaling. Mouse and human PD-1 share approximately 60% aa sequence identity (4).

1. Ishida, Y. et al. (1992) EMBO J. 11:3887.
2. Nishimura, H. and T. Honjo (2001) Trends in Immunol. 22:265.
3. Latchman, Y. et al. (2001) Nature Immun. 2:261.
4. Carreno, B.M. and M. Collins (2002) Annu. Rev. Immunol. 20:29.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for PD-1 Antibody (MAB1086)(9)

Publications using MAB1086

• R Motavalli, M Hosseini, MS Soltani-Za, A Karimi, M Sadeghi, S Dolati, M Yousefi, J Etemadi Evaluation of the immune checkpoint factors in idiopathic membranous nephropathy Molecular and cellular probes, 2023-04-21;69(0):101914. 2023-04-21 [PMID: 37075987] (Western Blot, Human)
• Nils Ludwig, Chang‐Sook Hong, Sonja Ludwig, Juliana H. Azambuja, Priyanka Sharma, Marie‐Nicole Theodoraki et al. Isolation and Analysis of Tumor‐Derived Exosomes Current Protocols in Immunology 12/1/2019 [PMID: 31763776]
• Nils Ludwig, Chang‐Sook Hong, Sonja Ludwig, Juliana H. Azambuja, Priyanka Sharma, Marie‐Nicole Theodoraki et al. Isolation and Analysis of Tumor‐Derived Exosomes Current Protocols in Immunology 2019-12-01 [PMID: 31763776] (Western Blot, Human)
• R Banga, C Rebecchini, FA Procopio, A Noto, O Munoz, K Ioannidou, C Fenwick, K Ohmiti, M Cavassini, JM Corpataux, L de Leval, G Pantaleo, M Perreau Lymph node migratory dendritic cells modulate HIV-1 transcription through PD-1 engagement PLoS Pathog., 2019-07-22;15(7):e1007918. 2019-07-22 [PMID: 31329640] (IHC-P, Human)
• Mingjing Shen, Yongbing Chen, Lijun Xu, Rongying Zhu, Xiang Xue, Ying Tsai et al. Increased infiltration of macrophages to radioresistant lung cancer cells contributes to the development of the additional resistance of tumor cells to the cytotoxic effects of NK cells International Journal of Oncology 5/4/2018 [PMID: 29750425]
• LJ Xu, Q Ma, J Zhu, J Li, BX Xue, J Gao, CY Sun, YC Zang, YB Zhou, DR Yang, YX Shan Combined inhibition of JAK1,2/Stat3?PD?L1 signaling pathway suppresses the immune escape of castration?resistant prostate cancer to NK cells in hypoxia Mol Med Rep, 2018-04-20;0(0):. 4/20/2018 [PMID: 29693186]
• Melendreras S, Martinez-Camblor P, Menendez A, Bravo-Mendoza C, Gonzalez-Vidal A, Coto E, Diaz-Corte C, Ruiz-Ortega M, Lopez-Larrea C, Suarez-Alvarez B Soluble co-signaling molecules predict long-term graft outcome in kidney-transplanted patients. PLoS ONE, 2014-12-05;9(12):e113396. 2014-12-05 [PMID: 25478957] (ELISA Development, Human)
• Daridon C, Loddenkemper C, Spieckermann S, Kuhl A, Salama A, Burmester G, Lipsky P, Dorner T Splenic proliferative lymphoid nodules distinct from germinal centers are sites of autoantigen stimulation in immune thrombocytopenia. Blood, 2012-09-06;120(25):5021-31. 2012-09-06 [PMID: 22955923] (IHC-P, Human)
• Sakthivel P, Ramanujam R, Wang XB, Pirskanen R, Lefvert AK Programmed Death-1: from gene to protein in autoimmune human myasthenia gravis. J. Neuroimmunol., 2007-11-26;193(1):149-55. 2007-11-26 [PMID: 18037500] (ELISA Development, Human)

Bioinformatics

• Gene Symbol: PDCD1
• Uniprot:
  • Q8IX89()