CEP-701 effects on FLT3/ITD-expressing cells. (A) Cells were incubated for 1 hour in culture medium with or without 50 nM CEP-701 and then lysed in detergent buffer. Then, 500 μg of this whole-cell extract was used for immunoprecipitation and immunoblotting as described in “Materials and methods.” The upper panel shows a blot probed with antiphosphotyrosine; the lower panel is the same blot stripped and reprobed with anti-FLT3. The blot reveals the characteristic appearance of FLT3, with a larger (160-kDa) mature, glycosylated form, and a smaller (130-kDa) immature form. (B) MTT assay of cells exposed for 48 hours to increasing doses of CEP-701. Each concentration point represents the mean of quadruplicate measurements of optical density (OD). The results are plotted as percent untreated control. This experiment was performed 3 times. The graph displays a representative result. Error bars represent the SD. (C) Annexin V apoptosis assay of cells exposed to CEP-701. The y-axis represents viable (ie, non-annexin V staining) cells plotted as percent untreated control. The concentration points represent a single measurement of annexin V binding. This experiment was repeated once with essentially identical results.