Skim milk solution

The halves of the hearts were homogenized, and total protein was extracted using protein lysis buffer (Pro-prep; iNtRON, Seongnam, Korea). Cardiac tissue samples containing 60 μg total protein and H9c2 cell extracts containing 10 μg total proteins were boiled for 10 minutes and loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (8% stacking and 10%, 15% separating gels). Separated proteins were transferred to nitrocellulose membranes (NC, 0.45 μm pore size; Bio-Rad, Hercules, CA, USA) or Iimmobilon-P transfer membrane (PVDF, 0.45 μm pore size; Millipore, Billerica, MD, USA). After blocking in 5% bovine serum albumin solution (Sigma-Aldrich) or 5% skim milk solution (BD Biosciences, San Diego, CA, USA) for 60 minutes, the membranes were incubated with primary antibody overnight at 4°C. The primary antibodies used are specified in Supplementary Table 1. Blots were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1:2,000; Jackson Immunoresearch, West Grove, IA, USA) or anti-mouse antibody (1:2,000; Jackson Immunoresearch) for 1 hour at room temperature. Glyceraldehyde-3-phosphate dehydrogenase was used as a protein loading control. Positive protein bands were visualized using an ECL kit (GenDEPOT, Barker, NY, USA), and results were quantified with an image analyzer (Image lab 3.0; Bio-Rad).