Mode of action of FF-10101. (A-B) Cells were treated with FF-10101 for the indicated hours, and cell cycle and apoptosis analyses were performed using BD Cycletest Plus DNA Reagent Kit and TACS annexin V Kit, respectively. (C-D) Cells were treated with FF-10101 in culture medium (C) or 100% human plasma (D) for 1 hour. Phospho- and total-FLT3 were detected by western blot analysis after immunoprecipitation with total-FLT3 antibody. Whole-cell lysates were also subjected to western blot analysis, for detection of phosphorylation status of downstream molecules of FLT3. (E) wild-type FLT3/FLT3-ITD–coexpressing 32D cells were preincubated with FLT3 inhibitors for 2 hours. After preincubation, 10 ng/mL of FL was added and further incubated for 20 minutes. Whole-cell lysates were subjected to western blot analysis for detection of phosphorylated and total FLT3 proteins.