Figure 4
In AML cells, FLT3-ITD can activate AKT, ERK, and STAT5 before it reaches the PM. (a–c) MOLM-14 (a, b), MV4-11, or Kasumi-6 cells (c) were treated with inhibitors of ER export (BFA or M-COPA) for 8 h. (a) MOLM-14 cells treated with 1 µM BFA (middle panels) or 1 µM M-COPA (bottom panels) were stained with anti-FLT3 (red) and calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (b) Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. (c) MV4-11 (left) or Kasumi-6 cells (right) were treated with M-COPA for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. Note that BFA and M-COPA inhibited the activation of AKT and ERK but not that of STAT5 through blocking FLT3-ITD trafficking from the ER to the Golgi apparatus. (d, e) MOLM-14 cells were treated with monensin (inhibitor of Golgi export) for 8 h. (d) Cells treated with 100 nM monensin were stained with anti-FLT3 (red) and lectin-HPA (Golgi marker, blue). Dashed line, cell border. Bars, 10 µm. (e) Lysates were immunoblotted with the indicated antibody. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. (f) MV4-11 (left) or Kasumi-6 cells (right) were treated with monensin for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 6.
