Chemiluminescence reagent

According to the standard protocol, 80 μg total protein samples were prepared and determined by BCA assay kit (KeyGEN). Then, 20 μg total proteins were separated by SDS-polyacrylamide gel, transferred to a PVDF membrane (KeyGEN), and blocked with 5% BSA in Tris-buffered saline containing 0.5% Tween 20, followed by incubation with primary (polyclonal rabbit-anti-rat SOD2 antibody, ZSGBBIO, China, 1 : 1000) and secondary antibody (goat-anti-rabbit antibody, ZSGBBIO, China, 1 : 8000). β-Actin (primary antibody, 1 : 200; secondary antibody, 1 : 6000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as an internal control. Protein bands were visualized using chemiluminescence reagent (KeyGEN).

Cellular extracts were lysed using the lysis buffer RIPA, which was purchased from KeyGen Biotech Co. Ltd (Nanjing, China), and supernatant was collected after centrifugation. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were blotted onto polyvinylidene difluoride membranes (Bio-Rad, USA). Then, membranes with isolated proteins were blocked for 1 h and detected using primary antibodies including anti-phosphor-ERK1/2 (MAPK1) (ab50011, pT185/pY187, 1:2000), anti-mTOR (ab2732, 1:2000), anti-phospho-mTOR (mTORC1) (ab137133, S2448, 1:1000), anti-ATG1 (ULK1) (ab167139, 1µg/ml), anti-LC3-II (LC3B) (ab48394, 1 µg/ml) and anti-β-actin (ab8227, 1:100) (Santa Cruz, USA) antibody at 4 °C overnight. After that, membranes were washed thrice by Tris-buffered saline with Tween 20 (TBST), and Goat Anti-Rabbit IgG H&L (HRP, 1:2000) were injected into the membranes, which were incubated for another 1 h. Finally, membranes were washed thrice again using TBST. Immunobinding signals were tested by the chemiluminescence reagent, which was purchased from KeyGen Biotech Co. Ltd. Relative protein expression was identified through densitometry analysis using the Image-Pro Plus Version 6.0 software and calculated based on the β-actin loading control.