Extraction and Analysis of Cardiac Proteins

Extraction of proteins from heart tissue samples and NRCM was performed as described previously [49 (link)]. in brief, heart tissue was homogenised and cell lysates were obtained in FAK lysis buffer (50 mM Tris-HCl, pH 7.5, 100 uM sodium pyrophosphate, 1 mM sodium fluoride, 150 mM NaCl, 0.1% SDS, 1% triton X-100, 0.5 mM EDTA) containing protease and phosphatase inhibitors. The lysates were obtained by centrifugation and the supernatants were subjected to immunoblotting analysis according to the manufacturers’ instructions. Each panel in each figure represents results from a single gel exposed for a uniform duration, with bands detected by LI-COR Odyssey imaging system (LI-COR, Inc., Nebraska USA). PPAR-γ and HMGCS2 (Abcam, Cambridge, MA). BDH1 and PDK4 (Thermofisher, Rockford, IL). Glut4 (Bioss, Woburn, MA), Bax, Histone 3, Cytochrome c, and Bad (Cell Signaling Technology, Danvers, MA). GAPDH (Millipore, Billerica, MA).