Cardiac Tissue Protein Analysis

Cardiac tissues were isolated from mice and stimulated with the indicated cardioplegia solutions for 1 hr. Lysates of cardiac tissue homogenates were obtained with lysis buffer (containing (mM) 150 NaCl, 20 Tris, 2 EDTA, 1% Triton X-100, and a protease inhibitor mixture) and were treated as previously described [18 (link)]. 30 μg denatured protein samples was subjected to SDS-PAGE. Proteins were visualized with NKCC1 (ab59191, Abcam), phosphoNKCC1 (#ABS1004, Millipore), Slc26a6 (ab172684, Abcam), CA IV (sc-74527, Santa Cruz Biotechnology), GAPDH (MA5-15738, Thermo Fisher), NHE1 (NBP1-76847, Novusbio), and β-actin (A3854, Sigma) antibodies using the enhanced luminescence (ECL) solution (Thermo Scientific). The intensity of protein band was normalized with that of β-actin as a protein loading control.