3.4. Western Blot

The total cellular proteins in each group were extracted by lysing with radioimmunoprecipitation assay buffer containing protease inhibitor (Sigma, USA). Protein concentration was measured by BCA Protein Assay Kit (Beyotime, China) and adjusted to the equal concentrations. Samples were boiled in SDS sample buffer for 10 minutes. For immunoblotting, each sample (containing 20 - 30 µg proteins) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 8% or 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto nitrocellulose membranes using wet transfer technology. For blocking, the membranes were incubated in Tris-buffered saline with 5% dry nonfat milk and 0.1% Tween-20 at room temperature for 1 hour. After washing by Tris-buffered saline with 0.1% Tween-20 (TBST), the membranes were probed with anti-TRPV5 antibody (Abcam, UK), anti-Vacuolar H+-ATPase (V-ATPase) antibody (Santa Cruz Biotechnology, USA), anti-TRAP, anti-Cathepsin K (CTSK) antibody (Abcam, UK), and anti-Carbonic anhydrase II (CA II) antibody (Abcam, UK). β-actin detected by β-actin antibody (Abcam, UK) was considered as a loading control. The expression of these proteins was visualized by a chemiluminescence kit (Merck Millipore, USA) after incubating with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Cell Signaling Technology, USA) by electro chemiluminescence (ECL) Tanon 5200 detection system (Shanghai, China). The density of the bands was determined using an Image Lab software (Bio-Rad Laboratories, USA).