The FLT3-840GS mutant is hyperphosphorylated and induces IL-3–independent growth in Ba/F3 cells.

(A) Ba/F3 cells transduced with either pMSCV-EYFP-IRES-FLT3-840GS or empty vector (pMSCV-EYFP-IRES, mock) were grown in the absence or presence of IL-3 as indicated. Results represent means ± SD of 3 independent experiments. (B) Lysates from Ba/F3 cells were immunoprecipitated with αFLT3 antibody and analyzed by Western blot using an antiphosphotyrosine (αPY) antibody, stripped, and reblotted with αFLT3 antibody. (C) Ba/F3 cells expressing either FLT3WT, FLT3ITD, FLT3-840GS, or empty vector (MIY) were incubated with a mouse isotype-matched control antibody (open histograms) or CD135-phycoerythrin (filled histograms) antibody. Viable cells were analyzed using a FACSCalibur flow cytometer. (D) Ba/F3 cells expressing FLT3WT, FLT3ITD, or FLT3-840GS were grown for 72 hours in the absence or presence of different concentrations of IL-3 as indicated. The growth of FLT3ITD-expressing Ba/F3 cells at 72 hours was defined as 100%. Results represent means ± SEM of 3 independent experiments.