Effect of leucine, insulin or in combination, on the phosphorylation state of p70S6K. Primary human myotubes were untreated or incubated in the presence of leucine (Leu; 5 mM), insulin (Ins; 100 nM) or co-treated with leucine and insulin (Leu + Ins) for 30 min (A), 3 h (B) or 24 h (C). Phosphorylation levels of p70S6K at Thr389 were determined by Western blot analysis relative to total levels of p70S6K. Data are presented as mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated.