Fig. 5. Effects of MAPK and PI3K inhibitors on the TPO-induced signaling pathway. (A) 2x10⁵ CD34⁺ cells were incubated overnight in serum-free medium starvation conditions, and then pre-treated for a brief period (20 minutes) with specific MAPK or PI3K pathway inhibitors followed by TPO incubation (40 minutes). The cells were then lyzed and the protein separated by SDS PAGE. The blots were probed with specific antibodies against phospho-ERK 1/2 (P-ERK 1/2), ERK 1/2, P-AKT, AKT, P-p70S6K (Thr389), P-p70S6K (Thr421/Ser424) or p70S6K. Representative immunoblots are shown. (B) The densitometric analysis of phosphorylated protein blots was performed with the chemidoc program Biorad, and expressed as fold increase of unstimulated cells, after normalization of total ERK, AKT and p706SK protein of each loading, and is presented as a bar graph. Results are expressed as the mean ± s.d. from three independent experiments.