P raptor s792

Protein extraction was carried out as follows: cardiac tissues were isolated, snap‐frozen and extracted in lysis buffer, and cardiomyocytes were harvested at the indicated time and extracted in lysis buffer. The lysis buffer was complemented with phenylmethanesulfonyl fluoride and phosphatase inhibitor. BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) was used to quantify total protein concentration. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk and incubated at 4°C overnight with the following primary antibodies: phospho‐adenosine monophosphate protein kinase (AMPK)‐T172, AMPK, p‐Raptor‐S792, Raptor, p‐mTOR‐S2448, mTOR, p‐p70S6K‐T389, p70S6K, β‐actin (1:1000; Cell Signaling Technology, Danvers, MA, USA) and caspase‐3 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was then incubated with the appropriate horseradish peroxidase‐conjugated secondary antibodies. The blots were developed using an enhanced chemiluminescence system.

Cucurbitacin E was obtained from Shanghai Shunbo Chempharm Co (Shanghai, China). Chloroquine (CQ), dimethylsulfoxide (DMSO) and sodium dodecyl sulfate (SDS), polyformaldehyde were from Sigma-Aldrich (St. Louis, MO, USA). WST-1 was from Roche (Penzberg, Germany). Lipofectamine RNAiMAX, Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, L-glutamine, and fetal bovine serum (FBS) were from Gibco/ Invitrogen (Carlsbad, CA, USA). Polyvinylidene difluoride (PVDF) membranes (Hybond-P) were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). Antibodies against p-p70S6K_T389, p70S6K, p-4E-BP1_T37/46, 4E-BP1, p-S6_S235/236, S6, p62/SQSTM1, LC3B, ATG5, Beclin 1, p-AKT_S473, p-Akt_T308, Akt, p-AMPKα_T172, AMPKα, p-AMPKβ1_S108, AMPKβ1, p-ULK1_S758, p-ULK1_S555, ULK1, p-Raptor_S792, Raptor, β-tubulin and HRP-conjugated second antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibody against LAMP2 was obtained from Abcam (Cambridge, MA, USA).