Ab32503

Rat atrium tissue lysate and whole cells were prepared by 1x cell lysis (Cell Signaling Technologies, USA) and 1x protease inhibitors (Cat. 04693159001; Roche Molecular Biochemicals, USA). The crude extracts were centrifuged at 13000×g, 4 °C for 10 min and the supernatant was collected. The insoluble sediment was resuspended in 8 mM guanidine hydrochloride in complete lysis. The protein concentration was measured by bicinchoninic acid protein assay and equal amounts of protein samples were separated on 12% polyacrylamide gels, transferred onto PVDF membrane. Membranes were blocked in 5% non-fat milk or 3% BSA at room temperature and incubated with primary antibodies overnight at 4 °C. The primary antibodies targeted against Caspase-3 (Cell Signaling Technology, 9662s), BCL-2 (Abcam, ab32124), BAX (Abcam, ab32503), Vinculin (Santa Cruz Biotechnology, sc-73614), LAMP-2A (Abcam, ab125068), LC3B (Abcam, ab192890), p62 (Abcam, ab109012), MYL4 (Abnova, HOOOO4635-M01), Cathepsin B (Abcam, ab214428) or Cathepsin L (Santa Cruz Biotechnology, sc-390367) at a 1:1000 dilution. After 1-h HRP-conjugated secondary antibodies incubation, bends were visualized using chemiluminescence (ECL, TANON, China) and viewed under Amersham Imager 600 system (GE Healthcare, USA). The relative intensity of each band was normalized to Vinculin.