Ex vivo bioassay for FLT3 inhibition. The immunoblot on the left was derived from peripheral blood leukemia cells isolated from a patient before and 1 hour after the initial dose of CEP-701. The blot on the right was obtained from TF/ITD cells exposed to the plasma from the same patient at the same time point. FLT3 was immunoprecipitated from detergent extracts of leukemia cells, resolved with SDS-PAGE, and transferred to PVDF membranes. The phosphorylated state of the receptor (upper bands, P-FLT3) was determined by immunoblotting with an antiphosphotyrosine antibody. Membranes were then stripped and reprobed with anti-FLT3 antibody to assess total FLT3 protein (lower bands, FLT3). Densitometric analysis of phosphorylated FLT3, normalized for the amount of total FLT3, is displayed graphically with columns.