Semi dry blot apparatus

Treated cells were washed with ice-cold PBS and lysed in homogenization buffer (10 mM Tris-HCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1:100 proteinase inhibitor cocktail) on ice for 30 min. After centrifugation for 30 min at 100,000× g at 4 °C to remove insoluble materials, the protein concentration of the lysate was determined using a BCA protein assay kit. Proteins were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); the resolved bands were electro-transferred to PVDF membranes using a semi-dry blot apparatus (Bio-Rad), and immunoblotting was performed by incubating PVDF membranes with 5% non-fat milk in Tris-buffered saline supplemented with Tween 20 (TBST, 10 mM Tris, pH 7.4, 150 mM NaCl, 0.2% Tween 20) for 1 h at room temperature to block residual free protein binding sites. The membrane was then incubated with different primary antibodies in 3% non-fat milk in TBST at 4 °C for 18 h. After repeated washing with TBST, the membrane was incubated with secondary antibodies conjugated with HRP. Immunoblots were developed using enhanced chemiluminescence, and the luminescence was visualized using a chemiluminescence detection system (Bio-Rad).