Effects of specific PTK inhibitors on KIT-transfected Ba/F3 cells. (A) Dose-response curves of the inhibitory activity of the PTK inhibitors imatinib, PKC412, and SU5614 in Ba/F3 KIT-WT and Ba/F3 KIT-D816V cells after 72 hours of incubation. Ba/F3 cells were seeded at a density of 0.05 × 106 cells/mL in the absence or presence of varying concentrations of imatinib, PKC412, and SU5614 and in the presence of 100 ng/mL recombinant human stem cell factor (SCF) in the case of KIT-WT. Viable cells were counted after 72 hours by trypan blue exclusion. The growth of untreated cells was defined as 100%. All values represent means and standard deviations from 3 independent experiments. (B) The 293 cells transfected with KIT-D816V were starved for 12 hours; treated with the indicated concentrations of imatinib, PKC412, and SU5614 for 2 hours; and lysed. Three hundred micrograms of each lysate was immunoprecipitated (IP) with α KIT antibody (α KIT) and immunoprecipitates were analyzed by Western blotting using antiphosphotyrosine antibody (αpTyr) and αKIT antibody.
