Deletion of wild-type R595 in FLT3-WT abrogates the ligand-dependent activation of FLT3-WT in Ba/F3 cells. (A) Schematic representation of wild-type R595 (FLT3-WT-ΔR595) deletion mutant in the FLT3-WT construct. (B) Ba/F3 cells expressing the FLT3-WT, FLT3-WT-ΔR595, FLT3-ITD-W51, and mock-transduced cells were seeded at a density of 4 × 10⁴ cells/mL in the absence or presence of IL-3 and FL (60 ng/ml). Viable cells were counted after 72 hours by trypan blue exclusion. The growth of cells with FL was defined as 100% (control). The standard error of the mean calculated from 3 independent experiments is indicated. (C) Western blot image showing the abrogation of activation of STAT5 in cells expressing FLT3-WT-ΔR595, when compared to FLT3-ITD-W51 and FLT3-WT, upon stimulation of FL (60 ng/mL). Cells were starved in the presence of 0.3% FBS for 24 hours. Blots were incubated with anti–phospho-STAT5 antibody, stripped, and reblotted with anti-STAT5 antibody.