CEP-701 potently and selectively inhibits FLT3 autophosphorylation.

(A) BaF3/ITD cells were incubated with increasing concentrations of CEP-701 for 1 hour. Whole cell detergent extracts (500 μg/sample) were prepared and immunoprecipitated with antihuman FLT3 antibody, followed by separation in 8% polyacrylamide gel electrophoresis and immunoblotting with the antiphosphotyrosine antibody, 4G10 (upper row). The membrane was stripped and reprobed with anti-FLT3 antibody to demonstrate equal loading of FLT3 in each lane (lower row). (B) BaF3 cells transfected with cDNAs for human PDGFR-β, FMS, and KIT were activated with their cognate ligand (PDGF, CSF-1, or KL) and treated with increasing concentrations of CEP-701 for 1 hour. Cells were lysed and immunoprecipitated with the corresponding antibody followed by immunoblotting with antiphosphotyrosine antibody 4G10 as above.