Gp130 antibody

The antibodies against p-STAT3^(Y705), STAT-3, p-NFκB-p65^(Ser276), NFκB-p65, p-Akt^(Ser473), Akt and CD44 (clone 156-3-c11) were from Cell Signaling Technology, Inc. (Beverly, MA, USA), gp130 antibody was purchased from R&D Systems (Minneapolis, MN, USA). Anti-human Notch-1 and EGFR antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-human-CD44-FITC, anti-human-CD24-PE, IgG2b-FITC, IgG1-PE antibodies and rhEGF were obtained from Immunotools (Friesoythe, Germany), and anti-Syndecan-1 (clone B-A38) was from Biorad (Hercules, CA, USA). Anti-human-Notch-2-PE & APC, Syndecan-1 (CD138)-PE antibodies were from eBioscience, Inc. (San Diego, CA, USA) and HRP–conjugated secondary antibodies were from KPL (Gaitherburg, MD, USA). Gamma-secretase inhibitor (GSI) was from Calbiochem (Darmstadt, Germany). Media, fetal calf serum (FCS) and tissue culture supplies were from Lonza (Basel, Switzerland). Unless otherwise stated, all chemicals were from Sigma (St. Louis, MO, USA).

An immortalized human podocyte cell line was cultured and maintained as described previously 5. Briefly, cells were routinely cultured in RPMI1640 medium supplemented with 10% foetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. Firstly, cells were incubated at 33°C for proliferation, and after reached at 70% confluence, the cells were transferred to 37°C for 2 weeks to allow differentiation. Differentiated podocytes were exposed to media containing high glucose (HG, final glucose concentration 30 mmol/l) or 19.9 mmol/l mannitol as osmotic control.
After individual pre‐treatment with gp130 antibody (2 μg/ml, R&D Systems, Minneapolis, MN, USA), IL‐6 antibody (1 μg/ml, R&D Systems, Minneapolis, MN, USA), recombinant sgp130 (1 μg/ml, R&D Systems, Minneapolis, MN, USA), recombinant human IL‐6 (20 ng/ml, Peprotech, Rocky Hill, NJ, USA) or complex of IL‐6 and recombinant human soluble IL‐6R protein (30 ng/ml, R&D Systems, Minneapolis, MN, USA), podocytes were exposed to HG or osmotic control for 24 hrs.