Palmitoylation of FLT3 at C563 specifically regulates FLT3-ITD–mediated signaling and cell growth. (A) Analysis of FLT3-ITD palmitoylation with the APE assay in 293T cells transiently expressing MigR1-FLT3-ITD. HAM, hydroxylamine (NH₂OH). (B) APE analysis of TF-1 cells stably expressing MigR1-FLT3-ITD treated with the palmitoylation inhibitor 2-BP or vehicle control ethanol (EtOH). (C-D) Examination of palmitoylation of endogenous FLT3 proteins in FLT3-ITD⁺ MV4;11 AML cell line (C) and FLT3-WT SEMK2 cells (D). (E) Palmitoylation status of FLT3-ITD mutants with various cysteines mutated to serines by using the APE assay. (F) Examination of the effect of C563S mutation on the palmitoylation of FLT3-WT and oncogenic FLT3-ITD and FLT3-D835Y mutants in 293T cells. (G) TF-1 cells stably expressing empty vector (MigR1 or EV), FLT3-ITD, or FLT3-ITD-C563S were established, and cell lysates were subjected to WB analysis for various signaling proteins. ITD-C563S proteins showed 2 bands in the immunoblot with long exposure (LE), in comparison with short exposure (SE). (H) Signaling sensitivity to human FLT3L (hFLT3L). TF-1 cells stably expressing different FLT3 variants were starved and stimulated with a graded concentration of FLT3L for 10 minutes. Cell lysates were subjected to WB analysis with the indicated antibodies. (I) TF-1 cells as in panel H were plated in triplicate in a graded concentration of FLT3L for 3 days. Cell growth was examined using the MTT assay. In all relevant panels, data are presented as means ± standard deviation. ***P < .001; ns, not significant, as determined by 2-tailed Student t tests. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.