2.5　Western blotting

Western blotting was used to assess expression of different proteins.MIN-6 cells were cultured in 6-well plates in one of the same five treatments as described in the previous section.After discarding the medium，cells were washed with PBS three times，then lysed in 200μL 2% Sodium dodecyl sulfate(SDS)per well.The extracts were warmed to 100℃for 10min and then mixed with 6×protein loading buffer(TransGen，J21020)and warmed to 100℃for 10min again.The extracts were separated by SDSPAGE(5% Stacking Gel，12%～15% Running Gel，70 V 30min，110 V 40min)，and then transferred to a polyvinylidene difluoride membrane(70 V 30～60min).After blocking with 5% nonfat milk in PBST(PBS plus 0.2% Tween-20)for 1h，the membrane was incubated with multiple primary antibodies：anti-LC3(MBL，PM036)，Anti-SQSTM1/p62(sequestosome 1)antibody(MBL，PM045)，Anti-S6K(ribosomal protein S6 kinase)antibody(CST，2708)，Anti-Phospho-S6K antibody(CST，9206)，Anti-AMPK antibody(CST，4811)，Anti-Phospho-AMPK(Thr 172)antibody(CST，4188)，Anti-procaspase-3 antibody(CST，9662S)，Anti-cleavedcaspase-3 antibody(CST，9661 T)，and Anti-GAPDH(glyceraldehyde-3-phosphate dehydrogenase)antibody(Yataihengxin，ZB002)at 4℃overnight.Recycling primary antibodies，washing with PBST three times，the membrane was incubated for 1h at room temperature with a secondary antibody of goat anti-mouse IgG1(Southern biotech，1070-05)or goat anti-rabbit IgG(Southern biotech，4050-05)，and then washed with PBST three times.ECL western blotting detection reagents(Pierce，32106)were used for the detection step.(https://www.daowen.com)