Effect of rapamycin on phosphorylation of p70S6kinase and 4E-BP1 in D816V cells. (A) Western blot analysis of HMC-1 (D816V) and α-155 (V560G) untreated or after exposure to rapamycin (5 nM) or imatinib (1 μM) as indicated. P indicates phosphorylated. (B) Kinetic effect of rapamycin on phospho–4E-BP1 on both cell types containing c-kit mutation. (C) D816V-transfected α-155 (V560G) cells were treated by rapamycin (5 nM), imatinib (1 μM), or both as indicated. (D) Effect of rapamycin action on wild-type BMMCs or BMMCs derived from D816V c-kit transgenic mice. Protein extracts were performed at the indicated time. Protein extracts were resolved by SDS-PAGE. Protein loading was assessed using polyclonal antibody against actin.