Disruption of palmitoylation alters FLT3-ITD intracellular localization. (A) Examination of FLT3 surface level elicited by C563S mutation. (B) Representative flow plots of surface FLT3 expression in TF-1 cells stably reconstituted with different MigR1-FLT3 variants. (C) Quantification of relative mean fluorescence intensity (MFI) of surface FLT3 by flow cytometry from 3 independent experiments. Data are presented as means ± standard error of the mean; P-values were determined by 2-tailed Student t tests. (D) Representative immunofluorescent confocal images of FLT3 (red) with the ER marker CANX (cyan, top panel), Golgi marker GM130 (cyan, middle panel), or AlexaFluor647-conjugated PM marker wheat germ agglutinin (WGA; bottom panel) in 293T cells expressing MigR1-FLT3-ITD or FLT3-ITD-C563S. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Bar represents 10 μm. (E) Quantification of percentages of cells expressing FLT3-ITD (n = 90) or FLT3-ITD-C563S mutant (n = 84) with FLT3 distribution predominantly in the ER or Golgi/PM, as shown in panel D. (F) Representative confocal images of 293T cells expressing MigR1-FLT3-ITD treated or not with 2-BP. Immunofluorescence staining was performed as that in panel D. Bar represents 10 μm. (G) Quantification of percentages of cells with FLT3-ITD distributed predominantly in the ER or Golgi/PM in the absence (n = 106) or presence (n = 120) of 2-BP, as shown in panel F. (E,G) P-values were determined by Fisher’s exact test; (C) 2-tailed Student t tests. In all relevant panels, *P < .05; ***P < .001; ns, not significant.