Human/Primate IL-6 Antibody

Detection of Recombinant Human IL‑6 by Western Blot. Western blot shows 25 ng of Recombinant Human IL-6 (Catalog # 206-IL), Recombinant Mouse IL-6 (Catalog # 406-ML) and Recombinant Rat IL-6 (Catalog # 506-RL). PVDF Membrane was probed with 1 µg/mL of Mouse Anti-Human/ Primate IL-6 Monoclonal Antibody (Catalog # MAB206) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for IL-6 at approximately 18 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 3.

IL‑6 in Human Skin. IL-6 was detected in immersion fixed frozen sections of hyperplastic human skin using Mouse Anti-Human/Primate IL-6 Monoclonal Antibody (Catalog # MAB206) at 8 µg/mL overnight at 4 °C. Tissue was stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections.

Cell Proliferation Induced by IL‑6 and Neutralization by Human IL‑6 Antibody. Recombinant Human IL-6 (Catalog # 206-IL) stimulates proliferation in the T1165.85.2.1 mouse plasmacytoma cell line in a dose-dependent manner (orange line). Proliferation elicited by Recombinant Human IL-6 (2.5 ng/mL) is neutralized (green line) by increasing concentrations of Mouse Anti-Human/Primate IL-6 Monoclonal Antibody (Catalog # MAB206). The ND₅₀ is typically 8.00 - 80.0 ng/mL.

Detection of Human IL-6 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 IL-6 by Western Blot Autocrine IL-6 activates STAT3 signalling in lung cancer cell-induced epidural ADSCs. a Epidural ADSCs were pre-treated with CM from lung cancer cells for 48 h, and pSTAT3 and STAT3 expression levels were detected by western blotting. Epidural ADSCs cultured in untreated medium served as a control. b The effects of lung cancer cell CM on epidural ADSC proliferation were evaluated using the CCK-8 assay. Epidural ADSCs were treated with CM from one of four lung cancer cell lines, and the optical density of both groups at 450 nm was analysed. Data from three separate experiments are shown. c Western blot analysis of pSTAT3 and STAT3 in epidural ADSCs treated with either 10 ng/mL recombinant IL-6, 10 ng/mL recombinant IL-11 or 50 ng/mL recombinant LIF in the presence or absence of either neutralizing antibodies or isotype controls. Loading control, actin. d Western blot analysis of pSTAT3 and alpha -SMA expression in epidural ADSCs treated with lung cancer cell CM in the presence or absence of neutralizing antibodies against IL-6, IL-11 or LIF. Loading control, actin. *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/31196220), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human IL-6 by Western Blot Autocrine IL-6 activates STAT3 signalling in lung cancer cell-induced epidural ADSCs. a Epidural ADSCs were pre-treated with CM from lung cancer cells for 48 h, and pSTAT3 and STAT3 expression levels were detected by western blotting. Epidural ADSCs cultured in untreated medium served as a control. b The effects of lung cancer cell CM on epidural ADSC proliferation were evaluated using the CCK-8 assay. Epidural ADSCs were treated with CM from one of four lung cancer cell lines, and the optical density of both groups at 450 nm was analysed. Data from three separate experiments are shown. c Western blot analysis of pSTAT3 and STAT3 in epidural ADSCs treated with either 10 ng/mL recombinant IL-6, 10 ng/mL recombinant IL-11 or 50 ng/mL recombinant LIF in the presence or absence of either neutralizing antibodies or isotype controls. Loading control, actin. d Western blot analysis of pSTAT3 and alpha -SMA expression in epidural ADSCs treated with lung cancer cell CM in the presence or absence of neutralizing antibodies against IL-6, IL-11 or LIF. Loading control, actin. *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/31196220), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human IL-6 by Western Blot Activated ADSCs trigger the proliferation and invasion of lung cancer cells by regulating MMP2/9 expression and EMT. a The effects of activated ADSCs on lung cancer cell proliferation were evaluated using the CCK-8 assay. Four lung cancer cell lines were cultured in the presence of ADSC-CM or aADSC-CM, and the optical density at 450 nm was analysed. The cancer cells cultured alone in normal growth medium were used as negative controls. Data from three separate experiments are shown. b The number of lung cancer cells that migrated through 8-μm Transwell membrane pores was counted to determine the changes in the invasive capabilities in response to CM from epidural ADSCs or aADSCs. c MMP2/9, E-cadherin and vimentin expression levels in four lung cancer cell lines treated with ADSC-CM or aADSC-CM were examined by western blotting. Lung cancer cells cultured in an untreated medium served as controls. d Lung cancer cells were treated with neutralizing antibodies against IL-6, isotype controls and aADSC-CM. Lung cancer cell invasion was analysed using a Transwell assay. Lung cancer cells cultured with untreated medium served as negative controls. e Lung cancer cells were treated with neutralizing antibodies against IL-6, isotype controls and aADSC-CM. MMP2/9, E-cadherin and vimentin expression levels in four lung cancer cell lines were analysed using western blotting. Lung cancer cells cultured with untreated medium served as negative controls. ***P < 0.001 Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31196220), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human/Primate IL-6 Antibody by Western Blot Musashi-1 mitigated DDP-induced apoptosis via the IL-6/AKT regulatory loopA. Cells were pretreated with 50 μM of LY294002 or vehicle for 3 hours, followed by 50 μM DDP treatment for 24 hours. The culture media were collected and the concentrations of IL-6 was determined by ELISA. B. 05MG-FlagMSI1 cells were treated with/without 50 μM DDP for 24 hours in the absence or presence of recombinant IL-6 (10 ng/ml) or anti-IL-6 neutralizing antibody (0.1 μg/ml). The cell lysates were analyzed by Western blot. C-D. The bar diagrams represent the quantified ratio of p-AKT-308/AKT and p-AKT-473/AKT obtained by the Western blot in B. E. 05MG cells were treated with control (serum free medium, S.F.) or condition media from 05MG-FlagMSI1 cells (C.M.) in the presence or absence of 50 μM DDP, IL-6 (10 ng/ml), and anti-IL-6 antibody (0.1 μg/ml) for 24 hours. The cell lysates were analyzed by Western blot to assess the levels of p-AKT-308, p-AKT-473, and total AKT. F-G. The bar diagrams represent the quantified ratio of p-AKT-308/AKT and p-AKT-473/AKT obtained in E. H. 05MG cells were treated as described in E and analyzed by Western blot. I-J. The bar diagrams represent the quantified ratio of cleavage-caspase-3/actin and cleaved-PARP/actin obtained in H. Data represent the mean ± S.D. of two independent experiments performed in triplicate. * P<0.05 vs AKT or Actin. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285760), licensed under a CC-BY license. Not internally tested by R&D Systems.