CEP-701 inhibits FLT3 phosphorylation in vivo.

Ten-week-old Balb/c mice were injected with 2 × 10⁶BaF3/ITD cells in PBS saline via tail vein on day 1. On day 12 at T = 0 hours the mice were given a subcutaneous dose of 20 mg/kg CEP-701. At 1, 2, 4, 8, and 12 hours after dosing, pairs of mice were killed, and the spleens (approximately 40-fold normal size by weight) were removed for assay. Splenic leukocytes were immediately harvested, lysed, and stored at −80°C until the next day, when immunoprecipitation with antihuman FLT3 and immunoblotting with antiphosphotyrosine antibody (500 μg/sample, upper panel) was performed. The blot was stripped and reprobed with the same anti-FLT3 antibody (lower panel). The antibody used for immunoprecipitation does not cross-react with mouse FLT3. For the control sample (“C”), the mouse was injected with vehicle only and killed 6 hours later.