2.4　Western blot analysis

MIN-6 cells were cultured in six-well plate.After discarding the medium，cells were washed with PBS three times，then lysed in 2% sodium dodecyl sulfate(SDS；200μL per well).The extracts were warmed to 100℃for 10min，then mixed with 6×protein loading buffer(TransGen；J21020)and warmed in 100℃for 10min.The extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE；4%stacking gel，12%～15% separating gel，70V for 30min，110V for 40min)，then transferred to nitrocellulose filter membrane(250mA for 30～60min).After blocking with 5%nonfat dry milk in PBST(PBS plus 0.2% Tween-20)for 1hr，the membrane was incubated with primary antibodies over-night in 4℃.After washing with PBST three times，the membrane was incubated with secondary antibodies(Goat Anti-Mouse IgG，SouthernBiotech，1070-05 and Goat Anti-Rabbit，SouthernBiotech，4050-05)1 hr at room temperature，then washed with PBST three times.The nitrocellulose filter membrane was incubated with ECL(SuperSignal West Dura，32106；Thermo Pierce)and imaged by Luminescent Image Analyzer(Model：ImageQuant LAS 4000 Mini，Serial No.3614294；GE Healthcare Bio-Sciences AB，Uppsala，Sweden).(https://www.daowen.com)