HCECs cultured on 33-mm culture dishes were lysed using lysis buffer containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, and 1 mM Na3VO4, pH 7.5, with a protease inhibitor mixture (1 mM PMSF, 1 mM benzamidine, 10 μg/mL leupeptin, and 10 μg/mL aprotinin) for at least 10 minutes Cells were scraped with a rubber policeman, followed by sonication (4 seconds by 4 cycles at 50 mV) and centrifugation (13,000 rpm for 15 minutes at 5°C). Supernatants were harvested and stored at −80°C until analysis. The protein concentration of each lysate was determined by bicinchoninic acid assay (micro BCA protein assay kit; Pierce Biotechnology, Rockford, IL). After boiling samples for 5 minutes, equal amounts of protein were fractionated onto 10% SDS-polyacrylamide gels, followed by electrophoresis and blotting onto polyvinylidine difluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked with blocking buffer, 5% fat-free milk in 0.1% Tris-buffered solution/Tween-20, for 1 hour at room temperature and then probed overnight at 5°C with antibodies of interest (1:1000). Membranes were incubated with goat anti-rabbit or mouse IgG for 1 hour at room temperature (1:2000). Immunobound antibody was visualized using an enhanced chemiluminescence detection system (ECL Plus; GE Healthcare, Piscataway, NJ). Images were analyzed by densitometry (SigmaScan Pro; Sigma). All experiments were repeated at least three times unless otherwise mentioned.