Western analysis

Rabbit polyclonal anti-Bcl-xl, rabbit polyclonal anti-Bad, rabbit polyclonal anti-cytochrome c. rabbit polyclonal anti-cleaved caspase-9, rabbit polyclonal anti-cleaved caspase-3, rabbit polyclonal anti-Akt and rabbit polyclonal anti-p-Akt, horseradish peroxidase labeled goat anti-rabbit immunoglobulin G (IgG), and rabbit anti-mouse IgG were purchased from Abcam (Abcam, Cambridge, MA, USA). Mouse anti-β-actin monoclonal antibody was obtained from Sigma (St. Louis, MO, USA). Frozen kidney tissue was homogenized for radioimmunoprecipitation. The homogenates were clarified by centrifugation at 12,000 g for 10 min at 4 °C, and the supernatants were collected. Supernatant samples (70 μg) were subjected to 15% polyacrylamide gel electrophoresis and transferred to cellulose acetate membranes. The membranes were blocked with 1 × casein solution for approximately 4 hours and then incubated with rabbit polyclonal anti-cleaved caspase-3 and anti-cleaved caspase-9, anti-Bcl-xl and anti-Bad, or anti-Akt and p-Akt antibodies overnight at 4 °C. The blots were washed with Tris-buffered saline-Tween-20 (TBST) buffer and subsequently incubated with goat anti-rabbit or rabbit anti-mouse IgG. After washing with TBST, the blots were developed with enhanced chemiluminescence reagents. A mouse monoclonal anti-β-actin antibody was used as a control for each sample. Signals of the blots were analyzed by the free image analyzing software Image J 1.42 (downloaded from http://rsbweb.nih.gov/ij/download.html).