Il 6rα

The 143B, HOS, and Saos-2 cells were treated with 0 ng/ml, 10 ng/ml, or 100 ng/ml of recombinant human IL-6 (PeproTech, Rocky Hill, New Jersey, USA) for 24 hours, and lysed using radioimmunoprecipitation (RIPA) buffer (Millipore, Temecula, California, USA) supplemented with a protease inhibitor cocktail, 0.5 mM phenylmethylsulfonyl fluoride, and 0.2 mM Na₃VO₄. The 143B, HOS, and Saos-2 cells were treated with 100 ng/ml of recombinant human IL-6 for two hours, followed by 0 μg/ml, 10 μg/ml, or 100 μg/ml tocilizumab for 24 hours and lysed using RIPA buffer supplemented with a protease inhibitor cocktail, 0.5 mM phenylmethylsulfonyl fluoride, and 0.2 mM Na₃VO₄. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the samples were adjusted to the same protein concentration before loading. Proteins were transferred to a nitrocellulose membrane and blotted using the following antibodies (at the dilutions recommended by the manufacturer): IL-6Rα (1:200 dilution; Santa Cruz Biotechnology, Dallas, Texas, USA), STAT3 (1:2000; Cell Signalling Technology, Beverly, Massachusetts, USA), and phospho-STAT3 (1:2000; Cell Signalling Technology). β-actin was assayed as the loading control.