Kidney Protein Extraction and Western Blot

Approximately 100 mg of frozen kidney was taken and pulverized to a fine powder, suspended in 900 µl of T-PER (Pierce, Rockford, IL, USA) containing 1 % protease and phosphatase inhibitors and homogenized. Supernants were collected by centrifugation (10,000×g for 10 min at 4 °C and the protein concentration determined using the 660 nm assay (Pierce, Rockford, IL, USA). Samples were equally diluted with 4× Laemilli buffer and then subjected to electrophoresis on 10 % PAGEr Gold Precast gel (Lonza, Rockland, ME, USA) before transfer to nitrocellulose membranes as detailed elsewhere [44 (link)]. Equal loading of protein was verified by Ponceau S staining of nitrocellulose membranes. Membranes were blocked with 5 % milk in TBST for 1 h and later probed with primary antibodies against p-stat-3 (Tyr 705), stat-3, cleaved caspase-3 and caspase-3 (Cell Signaling Technology, Danvers, MA, USA). After exposure to the primary antibodies, membranes were washed with TBST (3 × 5 min) and incubated with secondary anti-rabbit (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Immunoreactivity was visualized using Supersignal West Pico Chemiluminiscent substrate (Pierce, Rockford, IL, USA) and quantified by Fluorchem 9900 software (Protein Simple, Santa Clara, CA, USA).