Constitutive activation of PI-3K/Akt pathway in MO7e cells expressing Asp⁸¹⁶ mutant c-Kit.

(A) Different cells as indicated were incubated in 0.5% FCS in the absence of SCF for 5 hours and then stimulated with rhSCF (40 ng/mL) for 5 minutes. Akt protein was immunoprecipitated and the in vitro Akt kinase assay using GSK-3 fusion protein as substrate was carried out as described in “Materials and methods,” followed by Western blot analysis. The blot in the upper panel was probed with antiphospho–GSK-3 antibody. This blot was subsequently stripped and reprobed with anti-Akt antibody, as shown in the lower panel. (B) MO7e cells transduced with wild-type or D816H mutant c-kit were incubated in 0.5% FCS in the absence of SCF for 5 hours. LY294002 (20 μM) was added to some samples as indicated at the fourth hour of the incubation, followed by the experimental procedures as described in panel A.