4.11. Protein Synthesis Assay

The Surface Sensing of Translation (SUnSET) method was used for monitoring the global protein synthesis in the whole muscle. Puromycin, by structural analogy to aminoacyl-tRNAs, is incorporated into the nascent polypeptide and prevents the elongation; then, an anti-puromycin antibody is used to detect tagged proteins by immunoblot [82]. Dissected FDB muscles were stabilized for 2 h in DMEM 1% HS at 37 °C, as previously detailed. Then, the medium was replaced with a fresh one supplemented with 1 μM puromycin (EDM Millipore, Bedford, MA, USA), which was maintained for 45 min (pulse). The stimulation with exogenous ATP or mechanical perturbation started simultaneously with puromycin addition. When blockers or inhibitors were used, they were incubated 30 min before and during the puromycin. After the 45-min pulse, a 30-min chase was performed in DMEM 1% HS. Finally, muscles were processed for puromycin detection by immunoblot, as previously described. Densitometric analyses of immunoblot staining considered the entire lane. Either GAPDH signal or ponceau staining was used indistinctively for normalization.