Effect of IMC-EB10 on the phosphorylation of AKT and Stat5. Cells were incubated with varying amounts of antibody with or without exogenous FL (30 ng/mL). (A) In EOL-1 cells, AKT phosphorylation was up-regulated after FL stimulation (lanes 1 and 2 from right). Control antibody treatment did not affect AKT phosphorylation (lane 8 from right). In contrast, IMC-EB10 treatment (1-200 nM) completely inhibited FL-induced AKT phosphorylation. (B) In BaF3-ITD cells, constitutive phosphorylation of AKT was strongly inhibited with IMC-EB10 treatment (1-200 nM). (C) In EOL-1 cells, Stat5 was phosphorylated in the absence of FL stimulation (lane 1 from right), and FL stimulation did not significantly increase the level of phosphorylation of Stat5. Incubation with IMC-EB10 did not have any significant effect on Stat5 phosphorylation in this cell line. (D) In BaF3-ITD cells, constitutive phosphorylation of Stat5 was strongly inhibited with IMC-EB10 treatment (4-200 nM). In both cell lines, total AKT or Stat5 proteins were unaffected by antibody treatment.