Determination of Protein Synthesis by Puromycin Incorporation

To determine the rate of protein synthesis we utilized surface sensing of translation (SUnSET) methodology (Goodman et al., 2011). In vivo, 0.04 μmol/g puromycin (Calibiochem, Catalog #: 540222) was injected intraperitoneally into mice 30 min before harvest of skeletal muscle. Muscle was harvested at 0, 6, 24, 48 and 72 h after treatment and homogenized in Mueller’s Buffer (50 mM HEPES, 0.1% Triton-X100, 4 mM EGTA, 10 mM EDTA, 15 mM Na4P2O7, 100 mM β-glycerophosphate, 20 mM NaF, 5 mM NaVO4 and 1% protease inhibitor cocktail). In vitro, C2C12 myotubes were grown in 6-well plates. Puromycin was added into the cell culture medium (1 μM final concentration) exactly 30 min before harvesting the cells. Cells were scraped into ice-cold RIPA buffer (100 μl for one well of a 6-well plate) followed by ultrasound sonication on ice. Puromycin-containing proteins were analyzed by Western blot. Proteins were separated on 10% SDS-PAGE gels. Anti-puromycin antibody was purchased from Millipore (Catalog #: MABE343; Burlington, MA, United States).