Proteolysis assay

Proteolysis was measured as previously described (43), with the following modifications. Soleus and EDL muscles were isolated from 8-week-old female mice and preincubated in 1 ml of KRB buffer (in mM: 117 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 24.6 NaHCO₃, and 5 glucose) for 30 minutes with constant bubbling of 95% O₂/5% CO₂ before transferring to fresh 1 ml of KRB containing 0.5 mM cycloheximide to inhibit protein synthesis for 2 hours. Incubation buffer was collected, and tyrosine concentration was determined in 0.5 ml of incubation buffer as previously described (44) and was normalized to muscle weight.