Duplication of R595 in FLT3 induces IL-3–independent growth in Ba/F3 cells. (A) Localization of insertion mutants of FLT3 generated by duplication of 1 to 3 AAs of the stretch between 3 AA regions: 595 to 597, 589 to 591, and 602 to 604. (B) Ba/F3 cells stably transduced with FLT3-WT, FLT3-ITD-W51, FLT3-ins595R, FLT3-ins596RE, FLT3-ins597EY, FLT3-ins597REY, FLT3-WT-ins591YFY, FLT3-WT-ins602KWE, or mock-transduced cells were seeded at a density of 4 × 10⁴ cells/mL in the absence or presence of IL-3 or FL (60 ng/mL). Viable cells were counted after 72 hours by trypan blue exclusion. The growth of cells with IL-3 was defined as 100% (control). Standard error of the mean calculated from 3 independent experiments is shown. (C) Western blot showing the autoactivation of STAT5 in the mutants FLT3-ins595R, FLT3-ins597EY, FLT3-ins597REY, and FLT3-ITD-W51 when compared to FLT3-WT in unstimulated cells. FLT3-WT-ins595R, FLT3-WT-ins597EY, FLT3-WT-ins597REY, FLT3-WT-ins596RE, FLT3-ITD-W51, FLT3-WT, or mock-transduced cell lines were starved for 24 hours in the presence of 0.3% FBS and stimulated with 60 ng FL/mL for 5 minutes. Crude cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on a nitrocellulose membrane. Blots were then incubated with anti–phospho-STAT5 antibody, stripped, and reblotted with anti-STAT5 antibody. (D) Densitometric analysis of the Western image in panel C was used to quantify the ratio of phospho-STAT5 to total STAT5.