The following antibodies were used: LKB1 (#3047), AMPKα (#2603), AMPKαThr172 (#2535), mTOR (#2983), ULK1 (#8054), ULK1Ser757 (#14202), PI3Kinase Class III (#3358), and β-actin (#4970) (Cell Signaling Technology, USA). Beclin 1 and Bcl-2 (Santa Cruz Biotechnology, USA). LC3 (Novus Biologicals, USA), HRP tagged secondary antibody (Cell Signaling Technology, USA).
The myocardial tissues were homogenized in a buffer containing radioimmunoprecipitation assay cell lysate and the protease inhibitor phenylmethanesulfonyl fluoride. After homogenizing and centrifuging, the supernatant liquor was collected. The protein concentration was detected by bicinchonininc acid assay. The samples were heated at 100°C for 10 minutes. The proteins were separated using SDS-PAGE with 15% or 10%. Consequently, the protein bands were transferred to PVDF membranes. At room temperature, the membranes were blocked with 5% BSA solution for 1.5 hours and then incubated with the corresponding primary antibody (1 : 1000 dilution) at 4°C overnight. After the membranes were washed 5 times for 5 minutes with TBST, membranes were incubated with HRP-labeled secondary antibody (1 : 5000 dilution) at room temperature for 1 hour. The images were visualized and quantified by Tanon-5200 chemiluminescence imaging system (Tanon-5200 Multi, China).
The myocardial tissues were homogenized in a buffer containing radioimmunoprecipitation assay cell lysate and the protease inhibitor phenylmethanesulfonyl fluoride. After homogenizing and centrifuging, the supernatant liquor was collected. The protein concentration was detected by bicinchonininc acid assay. The samples were heated at 100°C for 10 minutes. The proteins were separated using SDS-PAGE with 15% or 10%. Consequently, the protein bands were transferred to PVDF membranes. At room temperature, the membranes were blocked with 5% BSA solution for 1.5 hours and then incubated with the corresponding primary antibody (1 : 1000 dilution) at 4°C overnight. After the membranes were washed 5 times for 5 minutes with TBST, membranes were incubated with HRP-labeled secondary antibody (1 : 5000 dilution) at room temperature for 1 hour. The images were visualized and quantified by Tanon-5200 chemiluminescence imaging system (Tanon-5200 Multi, China).