Smad7:β-catenin complex regulates myogenic gene transcription

a C2C12 myoblasts were cultured in growth media (GM) for 24？h, followed by differentiation media (DM) for designated times. Lysates were collected and assessed for the expression of muscle markers by western blot analysis. b Lysates were combined from several plates for immunoblotting detection of Smad7. Actin was used as loading control. c Co-localization of Smad7 and β-catenin was analyzed by immunofluorescence in ectopically expressing EYFP-Smad7-NLS and mcherry- β-catenin C2C12 cells. d Cytoplasmic and nuclear extraction was done to determine the endogenous localization of Smad7 and β-catenin in C2C12 cells. Extracellular signal-regulated kinase was utilized as cytoplasmic control and c-Jun as nuclear control. e Co-immunoprecipitation (Co-IP) assays were performed using Smad7 antibody to detect an interaction between endogenous β-catenin and Smad7 in extracts from mouse tibialis anterior (TA) muscle and C2C12 myoblasts. f Alternatively, in another separate experiment, C2C12 myoblasts were transiently transfected with Smad7-Flag. Smad7-Flag lysates were utilized for Co-IP with β-catenin or Flag antibody to detect interaction with β-catenin. IP with IgG served as control