Inhibition of Kit phosphorylation and Kit kinase activity by AP23464. (A) Inhibition of Kit phosphorylation by AP23464. Kit immunoprecipitates from cell lines expressing various Kit mutants were immunoblotted for phosphotyrosine (P-tyr) content in escalating doses of AP23464. Mo7e cells stimulated with SCF were used as a model of activated wild-type Kit. Immunoprecipitates were divided to simultaneously produce total Kit immunoblots as a loading control. Phospho-Kit levels were normalized for total Kit and 3 independent experiments were averaged to calculate IC₅₀ values and to generate the graph shown at bottom. Representative gels are shown for each cell line. (B) Inhibition of Kit kinase activity by AP23464. In vitro kinase assays of peptide substrate phosphorylation were performed with purified intracellular Kit or D816V Kit in escalating concentrations of AP23464. Graphs represent the mean reaction rates shown as percentages of the uninhibited reaction rate. Error bars depict standard deviation of the mean.