Mitochondrial Dynamics in Cardiomyocytes

Left ventricular cardiomyocytes were homogenized in ice-cold lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% triton X-100, 50 mM NaF, 5 mM Na₄P₂O₇, 1 mM Na₃VO₄, 1 mM DTT, and protease inhibitor cocktail) and protein content was determined using the BCA method (Thai et al., 2018 (link)). Briefly, 30 μg of protein was separated by 4–15% SDS-PAGE and transferred onto 0.2 μm supported nitrocellulose membrane using standard protocols. Blocked membranes were exposed to primary antibodies for PINK1 (Abcam ab23707), Parkin (Santa Cruz sc-32,282), mitofusin 2 (ab56889), DRP1 (Abcam ab56788), SQSTM1/p62 (Abcam ab56416), and LC3 (MLB, M186–3). GAPDH (Abcam ab9483) or VDAC1 (Abcam ab15895) were used as loading controls. Membranes were visualized using the Licor Odyssey infrared imaging system and analyzed with ImageJ software. Band intensities of all samples were normalized to an internal housekeeping protein that was included on each gel.