2.6. Western blot

Day 30^th vehicle- and T3+Dex-treated hiPSC-CMs were lysed with RIPA buffer supplemented with PhosSTOP and EDTA-free protease inhibitor cocktail (Sigma). All lysates were centrifuged at 13,000 rpm for 20 min at 4°C. Protein was quantified using Bradford Protein assay (Bio-rad). 22.5 μg of protein lysates were resolved on 4-20% Tris Glycine gel in Tris-Glycine-SDS buffer (Bio-rad) and transferred onto nitrocellulose membranes for immunoblotting. Membrane was blocked in TBST (Tris-buffered saline, 0.1% Tween 20) with 5% milk for 1 hour. Membranes were incubated with primary antibody anti-connexin 43 (1: 2,000, Abcam, ab217676) overnight at 4°C, and then in secorday antibody (1:2,000, Cell signal, #7074) for 1 hour. Membranes were incubated with ECL substrate (Thermo) for 5 min and developed on Bio-rad ChemiDoc MP imaging system (Bio-rad). After imaging, membranes were stripped with Restore Western Blot Stripping Buffer (Thermo), and stained with α-actinin (primary antibody: 1: 2,000, Sigma, EA-53; secondary antibody: 1:2,500, Invitrogen, 31430). Connexin 43 band intensities were quantified and normalized to α-actinin using Image J.