Lc3 1 2

Protein samples were extracted from the heart/cells as previously described [25 (link)]. For heart/cellular samples, proteins were lysed using the radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). After concentration determination using a bicinchoninic acid kit (Beyotime, Shanghai, China), proteins (80 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (12.5%) (EpiZyme, Shanghai, China), transferred to a nitrocellulose filter membrane (Millipore Corporation, Billerica, MA, USA), and blocked with 5% skimmed milk in PBS for 2 h. Samples were incubated in primary antibodies overnight at 4 °C, followed by incubation with fluorescence-labeled secondary antibody (1:10,000, Invitrogen, Carlsbad, CA, USA) for 1 h. Fluorescence was detected using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA). Primary antibodies against p62 (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat#5114S), LC3 I/II (1:500, Cell Signaling Technology, Cat#12741S), ATG4B (1:500, Sigma-Aldrich, Saint Louis, MO, USA, Cat#A2981), ATG3 (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat#3415S), and ATG7 (1:500, Cell Signaling Technology, Boston, MA, USA, Cat#2631S) were used. Glyceraldehyde 3-phosphate dehydrogenase (1:1000, Zhongshanjinqiao, Beijing, China, TA-08) was used as an internal control.