Fig 5. Serum induced activation of p70S6K independent of Insulin action.

Myoblasts were differentiated in low serum media, 2% FCS, containing DMEM-F12 GlutaMAX/1% ABAM for 5-d, forming myotubes. After 5-d myotubes were PBS washed (3 ×) and serum starved overnight in DMEM only (12 h) before exposure to DMEM-F12 GlutaMAX/1% ABAM alone (serum-free, DMEM only) or with the addition of 2% FCS, 2% CS-FCS or 2% HS containing 100nM Insulin, 100nM 1α,25(OH)₂D₃ or vehicle alone for 4 h and protein lysates collected. FCS and HS are sufficiently anabolic such that impact of Insulin treatment on p70S6K phosphorylation cannot be determined unless serum is removed, DMEM only. * P<0.05) from DMEM Control and Vitamin D. × exclusion of n = 1 outlier. For all other from n = 6 blots.