Pgsk3α β s21 s9

EB cell lysates were prepared using 1 × RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% Triton, 1% sodium deoxycholate and 0.1% SDS) supplemented with Complete Protease Inhibitor Cocktail (Roche) and PhosSTOP (Roche), and quantified with Bradford reagent (Sigma). Samples were prepared in Laemmli buffer (BioRad) and loaded on gels for SDS–polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinyl difluoride membrane (Millipore). The following primary antibodies were applied at the indicated dilution in Primary Antibody Signal Boost Immunoreaction Enhancer (Calbiochem); phosphorylated Smad1/5/8 (S465/8 and S426/8, 1:1,000, Cell Signaling), Smad1 (1:3,000, Abcam), cTnI (1:1,000, Abcam), β-active Catenin (1:1,000, Millipore), pGsk3α/β (S21/S9, 1:1,000, Cell Signaling). Gapdh (Abcam) and actin (Millipore) were diluted at 1:3,000 with 5% BSA in 1 × TBS–Tween 20. All primary antibodies were incubated for 12 h at 4 °C on a shaker. Enhanced chemiluminescence peroxidase-labelled anti-mouse and anti-rabbit antibodies (GE Biosciences) were diluted at 1:20,000 with 5% BSA in 1 × TBS–Tween 20. After washing the membranes, SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific) was utilized to detect the horseradish peroxidase signal. All uncropped western blots can be found in Supplementary Fig. 4.