Potent inhibition of FLT3 in various mutant cells. (A) The BaF3 cells expressing the indicated FLT3 mutation were incubated for 2 hours with the indicated concentrations of G-749, and (B-C) for direct comparison with 3 inhibitors, the BaF3 cells expressing FLT3-ITD/F691L (B) or FLT3-D835Y (C) were incubated with the indicated concentrations of G-749, AC220, and PKC412 for 2 hours. The autophosphorylation level of FLT3 was visualized and compared by western blotting analysis. (D) Molm-14 cells were treated with FLT3 inhibitors in the presence of 5 ng/mL of FL for 2 hours. The autophosphorylation level of FLT3 was then analyzed. (E) For direct comparison, the phosphorylation levels of FLT3, STAT5, ERK1/2, and AKT were analyzed by immunoblotting as in panel D. Total protein of each protein was used as a loading control, otherwise specified.