Med12

Following a NC, PTU, or PTU + T3 diet for 4 or 16 weeks, hearts were harvested, rinsed in cold phosphate buffered saline, and flash frozen. Cardiac ventricles were isolated and homogenized in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS; Thermo) containing cOmplete Mini protease inhibitors (Roche). Protein lysate concentrations were quantified by BCA assays, and 40 μg of protein was separated by 7.5% SDS-PAGE gels. Proteins were transferred to 0.45 μm nitrocellulose (Biorad) at 35 V for 16 h at 4° C and stained with ponceauS. Blots were blocked for 1 h at room temperature in 3% Bovine Serum Albumin/1% Polyvinylpyrrolidone and incubated overnight at 4°C with primary antibodies to detect Med12 (Cell Signaling cat#4529, 1:1000), Med13 (Bethyl cat#A301–277A, 1:500), CDK8 (Santa Cruz cat#sc-1521, 1:1000), Med1 (Bethyl cat#A300–793A, 1:1000), and GAPDH (Cell signaling cat#2118, 1:3000). Blots were incubated for 2 h with HRP-conjugated secondary antibody and developed with ECL reagents (GE healthcare) using chemiluminescent-sensitive X-ray film (Fisher Scientific).