Cells were harvested, washed twice, and then resuspended with lysis buffer. After being denatured with SDS loading buffer by heating, equal amount of protein sample was uploaded and separated with SDS–polyacrylamide gel electrophoresis (PAGE). Then, the proteins were transferred from a PAGE to the polyvinylidene difluoride membrane. After being blocked with 5% fat-free milk, membranes were incubated with primary antibodies at 4°C overnight. The second day, membranes were washed three times with Tris-Buffered Saline Tween-20 and then incubated with horseradish peroxidase–conjugated secondary antibodies at room temperature for about 2 hours. After a second round of washes, membranes were treated with enhanced chemiluminescence reaction buffer, and each band was visualized.

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