Gdf 8

All animal studies were performed as approved by the Harvard Committee on Animals. Adult (1-year-old) C57Bl/6 male mice were obtained from Charles River, and intravenously injected (by tail vein injection) with 0.5 mg/kg GDF11 (PeproTech) or 0.5 mg/kg, 1 mg/kg, 2 mg/kg and 4 mg/kg GDF8 (R&D Systems) or saline as control. Ligands were reconstituted in water at a concentration of 1 mg/mL and diluted in saline prior to injection. Heart tissue was collected 1 h post injection. Whole heart protein lysates were obtained by homogenizing the heart in RIPA buffer freshly supplemented with 1 mM PMSF and protein phosphatase inhibitor 2 and 3 (Sigma-Aldrich). 40 g total protein was loaded in NuPAGE 4-12% Bis-Tris gels (LifeTechnologies). Following transfer, membranes were blocked with non-fat dry milk for 1 h at room temperature and successively incubated with primary pSMAD2 antibody (Millipore, Cat. no. AB3849; Lot 2649232) and total SMAD2/3 antibody (Cell Signaling Technology, Cat. no. 8685P; Lot 4) overnight at 4 °C. Proteins were detected with horseradish peroxidase-conjugated antibodies (BioRad Laboratories; Cat. no. 172-1019; Lot L006328 A) and enhanced chemiluminescence (Amersham™ GE Healthcare, Cat. no. RPN2236).