2.4　Western blot analysis

Briefly，the cells were lysed with 2% SDS.The extracts were heated to 100℃for 10min，then mixed with 6×protein loading buffer(TransGen，Beijing，China)and heated to 100℃again for 10min.After separation by 10%，12% or 15% SDS-PAGE，the extracts were transferred to a polyvinylidene difluoride membrane.After being blocked with 5% milk in Tris-buffered saline containing 0.1% Tween for 1h，the membrane was incubated with primary antibodies，washed with PBST three times，and then incubated with secondary antibodies.After three times wash with PBST，the membrane was incubated with ECL western blot substrate(SuperSignal West Dura；Thermo Pierce，Rockford，USA)and imaged using a Luminescent Image Analyzer(GE Healthcare Bio-Sciences，Uppsala，Sweden).(https://www.daowen.com)