4.6. Western Blot Analysis

Cells were harvested, lysed, and an equivalent amount of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes for immunoblotting. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline for 1 h with a Tween-20 buffer (TBS-T; 20 mM Tris, 100 mM NaCl, pH 7.5, and 0.1% Tween-20) at room temperature. Membranes were then incubated with primary antibodies overnight at 4 °C. The membranes were washed with a TBS-T buffer and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h. The membranes were washed with TBS-T buffer. Antigen-antibody complexes were detected using an enhanced chemiluminescence detection system (GE Healthcare, Chicago, IL, USA).