Cardiac Protein Extraction and Western Blot Analysis

Th total protein extracts were prepared from 10 to 15 mg of the deep-frozen heart tissue. To maintain the extract integrity and function, we used Complete Protease Inhibitor Cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA), Phosphatase Inhibitor Cocktail II (ab201113, Abcam, Waltham, MA, USA), PMSF (1 mM), EGTA (1 mM), and EDTA (1 mM). The proteins were isolated using a 1xRIPA buffer (ab156034, Abcam, Waltham, MA, USA). The concentration of the protein was determined by the Lowry method [15 (link)]. The samples (30–50 µg) were diluted in Laemmli buffer, separated by 12.5% SDS-PAGE, and transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Wilmington, USA) [12 (link)]. The relative levels of the detected proteins were visualized using a C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, Nebraska, USA) and normalized to the appropriate loading control. The antibodies used: (ab56788) anti-DRP1 (1:1000), (ab119685) anti-OPA (1:1000), (ab124773) anti-Mitofusin2 (1:1000), (ab54481) anti-PGC1a (1:1000), (cs2132) anti-Parkin (1:1000), (Affinity Biosciences, DF7742) anti-PINK1 (1:250), (Affinity Biosciences, AF5384) anti-SQSTM1/p62 (1:250), (Affinity Biosciences, DF8163) anti-BNIP3L (1:250), and (cs12741) anti-LC3A/B (1:1500). The anti-GAPDH (ab181602) and anti-tubulin (1:2000) (ab4074) antibodies were used as a loading control.