Phosphorylation of STAT5 and ERK, downstream effectors of FLT3, is restored by the addition of IL-3 even after incubation with a high concentration of MLN518. Ba/F3 cells stably expressing FLT3-ITD or FLT3 activation loop mutations were incubated in 10 μM MLN518 for 30 minutes, with or without the addition of IL-3 for 5 minutes at the end of the incubation, as indicated. Whole-cell lysates (WCL) were prepared, resolved by SDS-PAGE, then immunoblotted with antiphospho-STAT5, anti-STAT5, anti-phospho-ERK, or anti-ERK antibodies, as indicated.