Effect of rapamycin on total protein degradation. Myotubes were incubated with 0.2 × PC AA (filled bars) or 1.0 × PC AA (open bars) media (A), or with 30 (filled bars) and 750 (hatched bars) μM leucine in 0.2 × PC AA medium (B). After labelling C2C12 myotubes for 48 hours, cells were incubated with 0.2 × PC AA or 1.0 × PC AA for two hours. Rapamycin (RAP) 2.2 × 10-8 M was then added to half of the wells containing 0.2 × PC AA or 1.0 × PC AA and dishes transferred back to incubator. Twenty minutes later insulin 10-8 M was added in to half of the rapamycin treated and half of the untreated cells and incubated at 37°C for another 8 hours. Since rapamycin has half-life of about 3–4 hours media was supplemented with rapamycin after the first four hours of incubation. Data represent mean ± SEM from one or two experiments performed in triplicates. * Indicates differences between two amino acids concentrations, ψ represents rapamycin treated versus non-rapamycin treated (control) group and # symbolises difference between insulin and rapamycin treated versus insulin treated groups.