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Western blot analyses Whole-cell heart tissue lysates were prepared by homogenizing flash-frozen hearts in ice-cold Tris-HCl buffer (pH 7.5) containing 0.5% Triton-X 100, 4 mM EGTA, 10 mM ethylenediaminetetraacetic acid (EDTA), 150 mM sodium chloride, 1 mM sodium orthovanadate, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, and a complete mixture of protease inhibitor cocktail 32,35,38