Enhanced chemiluminiscence kit

Western blot analysis was performed according to the standard protocol [46] (link). Briefly, the cells were solubilized in lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X100, 0.5% NP40, 25 µg/mL each of aprotinin, leupeptin and pepstatin, 1× phosphatase inhibitor cocktail (Sigma). After clarification at 10,000 g for 30 min, the supernatant was used for protein analysis. Protein concentration determined by the Bio-Rad protein assay kit ((Bio-Rad, Hercules, CA) and aliquots containing 25 µg protein were separated by SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting and subjected to western blot analysis with the recommended dilution of primary antibody. Following washes the blots were reacted with secondary antibody mix containing 1∶2500 dilutions of the appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). Protein bands were visualized using a commercially available enhanced chemiluminiscence kit (Amersham Biosciences). Blots were also immuno-reacted with a 1∶5000 dilution of anti-actin mouse monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) to normalize for variation in protein loading.