Ma1 83347

According to previous studies, total protein was extracted from rat myocardial tissue and cultured cardiac cells with RIPA lysis buffer supplemented by a protease inhibitor cocktail (Complete, Roche). After quantification using the BCA kit (Beyotime Institute of Biotechnology, China), the equal amount of protein samples were separated by SDS-PAGE and then transferred to the PVDF membrane. The membranes were blocked with nonfat milk in 1X TBST for 1 hour at room temperature and then incubated with the primary antibodies at 4°C overnight. The next day, the membranes were washed using 1 X TBST for 3 × 5 min, and then, the membranes were incubated with secondary antibodies (at an optimized concentration) for 1-2 hours at room temperature. Finally, an image analyzer (Bio-Rad) was used to detect and evaluate the band density on the film. The primary antibodies include against RIP3 (PRS2283, Sigma), β-MHC (MA1-83347, ThermoFisher Scientific), ANP (PA5-29559, ThermoFisher Scientific), MLKL, ZO-1 (40-2300, ThermoFisher Scientific), GAPDH (PA1-987, ThermoFisher Scientific), and ACTIN (ab6276, Abcam). The secondary antibody used including goat anti-rabbit IgG and goat anti-mouse IgG was purchased from Abcam (ab6789, ab205718, Abcam).