Figure 3
FLT3-ITD retention in the Golgi region is dependent on its tyrosine kinase activity. (a, b) MOLM-14 cells were treated for 4 h with AC220 (a) or PKC412 (b). Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. Full length blots are presented in Supplementary Fig. 5. (c) MOLM-14 cells were treated with AC220 (upper graph) or PKC412 (lower graph) for 48 h. Cell proliferation was assessed by ATP production. Results are means ± s.d. (n = 3). (d) Lysates from MOLM-14 were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (endo H) then immunoblotted with anti-FLT3 antibody. CG complex-glycosylated form, HM high mannose form, DG deglycosylated form. Full length blots are presented in Supplementary Fig. 5. (e, f) MOLM-14 cells were treated with 10 nM AC220 or 100 nM PKC412 for 8 h (e) or 16 h (f). (e) Fixed cells were permeabilized, then immunostained with anti-FLT3 (red) and anti-calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (f) Non-permeabilized cells were immunostained with an anti-FLT3 extracellular domain (ECD) antibody. Bars, 10 µm. Note that FLT3 tyrosine kinase inhibitors inactivated FLT3, then released the receptor from the Golgi region for localization to the PM.
