IMC-EB10 inhibits FL-induced phosphorylation of wild-type FLT3 and ligand-independent constitutive phosphorylation of FLT3-ITD. (A) EOL-1 and (B) EM3 cells were incubated in varying concentrations of antibody in the presence or absence of FL (30 ng/mL). Lanes 1 and 2 (from right) indicate that a low level of FLT3 phosphorylation existed in untreated EOL-1 cells and that ligand strongly increased the level of receptor phosphorylation. Ligand-induced receptor phosphorylation was blocked by IMC-EB10 antibody but not by a control antibody (control, IMC-C225 at 200 nM). (C) BaF3-ITD and (D) MV4;11 cells were incubated in varying concentrations of antibody without exogenous FL. Ligand-independent phosphorylation was inhibited by IMC-EB10 treatment. Total FLT3 protein was unaffected by antibody treatment.