| Cells were lysed at 4°C in buffer containing 1% SDS, 10 mM Tris-HCl pH 7.4, supplemented with protease inhibitors (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (Sigma Chemical Co., St. Louis, MO). Viscosity of the samples was reduced by brief sonication. 40 µg of protein (Bio-Rad Protein Assay) were boiled for 5 min in Tris-glycine-SDS sample buffer (Invitrogen) and heated at 70°C for 10 min, then separated by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). |
After blocking nonspecific binding sites for 1 h with 5% milk in TPBS (phosphate-buffered saline, Tween20 0.1%), membranes were incubated overnight with primary antibody. After three washes in TPBS, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit (1∶10,000 dilution), goat anti-rabbit (10,000 dilution), or anti-mouse (1∶5,000 dilution) antibody (Amersham Biosciences, Piscataway, NJ) for 1 h and then washed three times in TPBS. |
Immunoblots were revealed using enhanced chemiluminescence detection kit (Pierce) by autoradiography. |