Western blot analysis

Western blotting was used to measure the apelin, APJ, Bcl-2, and Bax expression levels in the left ventricular tissues. The frozen tissues were weighed and homogenized in RIPA lysis buffer. Protein levels in the supernatant were determined using the Bradford method (21). Thereafter, 40 µg of protein was subjected to SDS-PAGE with 10% separation and transferred onto a nitrocellulose membrane for 3 h at 200 mA. The membranes were blocked with 1% bovine serum albumin for 1 h. Subsequently, primary antibodies for apelin (ab125213, dilution, 1:500; Abcam,

Cambridge, United Kingdom), APJ (ab214369, dilution, 1:500; Abcam, Cambridge, United Kingdom), Bcl-2 (ab59348, dilution, 1:500; Abcam, Cambridge, United Kingdom), and Bax (ab53154, dilution, 1:500; Abcam, Cambridge, United Kingdom) were added, followed by overnight incubation at 4°C. After washing twice with TBS, membranes were incubated with the respective secondary antibodies (dilution, 1:10,000; Santa Cruz, CA, USA). Finally, protein band intensities were quantified using a densitometer analysis system (Quantity One software, Bio-Rad, PA, USA).