Chemidoc 2000

Radioimmunoprecipitation assay buffer extraction buffer (50 mM Tris–HCL pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1% NP-40, 0.2% SDS, 1 tablet Roche Complete MINI protease inhibitor, pH 7.5) was added to cell culture plates, scraped, collected, and kept on ice for 45 min with vortexing every 15 min. Lysates were spun at 4°C for 30 min at 5,800 × g. 25 μL of concentrated supernatant was treated with 40 μL of 0.225 mM Alexa 647 C2-maleimide and incubated for 2 h at room temperature in the dark, with rotation. 450 μL of ice-cold acetone was added and incubated for 1 h at −20°C. The protein pellet was resuspended in 1× Laemmli buffer (#161-0737, BioRad, Hercules, CA, United States) and loaded into a 4–15% TGX gel (#4561086, BioRad, Hercules, CA, United States). Gels were imaged with ChemiDoc-2000 (BioRad, Hercules, CA, United States) using the Alexa 647 preset. Protein was then transferred onto PVDF membranes and probed with anti-RyR 1:1000 (#MA3-916, Thermo Fisher, Waltham, MA, United States) and donkey anti-mouse secondary antibody (#PA1-28748, Thermo Fisher, Waltham, MA, United States). Blots were developed as described above.

Protein expression was determined using the following antibodies in immunoblotting: p62 1:1000 (Abcam #ab56416), PINK1 1:1000 (Cell Signaling #9646), p53 (Abcam #ab17869), LC3 (Abgent # AM1800A), DRP1 1:500 (Abcam #AB56788), and MFN2 1:500 (Sigma #WH0009927M3). Left ventricular free-wall tissue was homogenized in protein lysis buffer (50 mM Tris–HCL pH 7.5, 150 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1 mM Phenylmethylsulfonyl fluoride, 1 tablet Roche Complete MINI protease inhibitor). Samples were centrifuged at 2.5 k RPM for 15 min, aliquoted, and stored at −80°C. A total of 15–25 μg protein was loaded into 4–15% TGX gels (#4561086, BioRad, Hercules, CA, United States) via SDS-PAGE, transferred onto Polyvinylidene difluoride (PVDF) membranes, and probed with mouse or rabbit antibodies specific for these proteins and subsequently probed with donkey anti-mouse or anti-rabbit secondary antibodies (Thermo Fisher, Waltham, MA, United States). Blots were developed with SuperSignal West Pico (#34080, Thermo Fisher, Waltham, MA, United States), imaged with ChemiDoc 2000 (BioRad, Hercules, CA, United States), and quantified and analyzed using ImageLab (BioRad, Hercules, CA, United States) and ImageJ (US National Institutes of Health, Bethesda, MD, United States) softwares.