Alkaline phosphatase conjugated anti rabbit igg

Cells were pre-treated with 10 ng/ml human recombinant IL-6, 40 µg/ml human IL-6R neutralizing antibody or 60 µg/ml human gp130 neutralizing antibody for 24 h, following by 5 µM cisplatin treatment for 6 h. For the stimulation assay, cells were treated with 1 or 10 ng/ml IL-6 for 30 min. Cells were harvested and lysed in CelLytic M Cell Lysis reagent (Sigma-Aldrich) with protease and phosphatase inhibitor cocktails (Pierce Biotechnology, Rockford, IL, USA). Protein concentrations were determined (Bio-Rad, Munich, Germany), and 50 µg proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto PVDF membranes (Bio-Rad). After blocked with 5% BSA for 1 h, the membranes were incubated with primary antibody to human anti-phospho-STAT3(Tyr705) (rabbit polyclonal, 1:1,000; Cell Signaling Technology, Beverly, MA, USA), anti-STAT3 (mouse monoclonal, 1:5,000; R&D Systems) and anti-GAPDH (mouse monoclonal, 1:1,000; Abcam, Cambridge, UK) overnight at 4°C. The blots were then washed three times and incubated with alkaline phosphatase-conjugated anti-rabbit IgG (1:10,000; Sigma-Aldrich) or mouse IgG (1:10,000; Dako, Glostrup, Denmark) antibodies at room temperature for 1 h, then washed three times and visualized with ECF substrate in a scanner (Storm) (both from GE Healthcare, Uppsala, Sweden).