The positive charge of duplicated arginine 595 is critical for the transforming potential of FLT3-ITDs. (A) Shown are 3 different substitution and 2 deletion mutants of duplicated R595 generated in 2 different FLT3-ITDs (W51 and NPOS). The deletion mutant of duplicated E596 in FLT3-ITD-W51 is also shown. (B) Ba/F3 cells stably transduced with FLT3-WT, FLT3-ITD-W51, FLT3-W51-R602A, FLT3-W51-R602E, FLT3-W51-ΔR602, FLT3-W51-ΔE603, or mock-transduced cells were seeded at a density of 4 × 10⁴ cells/mL in the absence or presence of IL-3. Viable cells were counted after 72 hours. The growth of cells with IL-3 was defined as 100% (control). Standard error of the mean calculated from 3 independent experiments is indicated. (C,D) The mutants FLT3-NPOSΔR623 and FLT3-W51-R602K were expressed in Ba/F3 cells and analyzed as described in panel B.