Ripa p1003b

H/R-induced H9c2 cells were treated with SXD, and then the proteins from each treatment group were isolated by radio immunoprecipitation assay buffer (RIPA, P1003B, Beyotime, China). The supernatant was centrifuged at 14,000 rpm for 15 min at 4 °C, and the total protein concentration was determined, followed by quantitation with the bicinchoninic acid assay (BCA) protein assay kit (P0010, Beyotime, China). After denaturation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, the equal amounts of proteins were transferred to nitrocellulose membrane (HATF00010, Millipore, USA). Blots were blocked with 5 % fat-free milk in TBS with Tween-20 (TBST) at room temperature (approximately 25 °C) for 1 h, and then Caspase3 (1:500, 19677-1-AP, Proteintech, USA), Caspase9 (1:500, bs-20773R, Bioss, Switzerland), Cytochrome C (1:500, bs-0013R, Bioss, Switzerland), β-actin (1:5000, 200068-8F10, ZEN-BIOSCIENCE, China) primary antibodies were added in 5 % blocking buffer at 4 °C overnight. After incubation with the corresponding HRP-conjugated secondary antibodies (1:2000, SA00001-1 and SA00001-2, Proteintech, USA), the enhanced chemiluminescence (ECL) kit (WBKLS0500, Millipore, USA) was used for detection. The grey scale values of protein bands were analyzed using Image J software (NIH, USA).