2.8. Western blot analysis

Protein was extracted by lysis for 30 minutes on ice in RIPA buffer (Cell Signaling). Proteins were separated in SDS‐PAGE and then transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% skim milk in TBS‐T (150 mmol/L NaCl, 10 mmol/L TRIS, PH 7.6 and 0.1% TWEEN‐20) at room temperature for 1 hour, washed for 5 minutes 3 times with TBS‐T and then incubated with primary antibodies at 4°C overnight. All the primary antibodies were obtained from Cell Signaling Technology. After that, membranes were then washed with TBS‐T for 10 minutes 3 times and incubated with appropriate HRP‐conjugated secondary antibody at room temperature for 1 hour. Ultimately, the proteins were measured using a Gel Doc 2000 (Bio‐Rad).