Figure 2: Kit(D814Y) localizes to endolysosomes in mouse RCM cells.

(a) Glycosylation of Kit(D814Y). Lysates from pt18, R and RCM cells were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (endo H), then immunoblotted with anti-Kit. CG, complex-glycosylated form; DG, deglycosylated form; HM, high mannose form. NB: most Kit(D814Y) was a mature complex-glycosylated form. (b) Localization of normal Kit and Kit(D814Y). RCM, R or pt18 cells were fixed with PFA and stained with anti-Kit. Phase contrast images are shown. Bars, 10 μm. NB: normal Kit accumulated at the PM; Kit(D814Y) at vesicular structures. (c–g) Localization of Kit(D814Y) to endolysosomes. (c) PFA-fixed or (d) methanol-fixed RCM cells were double-stained with anti-Kit (green) in conjunction with the indicated antibody (red). Insets show the magnified images of the boxed area. Representative images of Kit-positive endolysosomes containing cathepsin D are shown. Bars, 5 μm. CD63, cluster of differentiation 63; EEA1, early endosome antigen-1; LAMP1, lysosome-associated membrane protein-1; TGN46, trans-Golgi network 46. (e) Endolysosomal localization of Kit(D814Y)-GFP. RCM cells transfected with Kit(D814Y)-GFP. (Upper panels) Cells were stained with anti-LAMP1 (endolysosome marker; red). (Lower panels) For visualizing endocytic compartments, cells were incubated with 1 mg ml⁻¹ AF647-dextran for 1 h. Expressed protein and dextran were visualized by GFP (green) and AF647 fluorescence (blue). Magnified images of the boxed area are shown. Arrowheads indicate Kit-positive endolysosomes that contain dextran. Bars, 5 μm. NB: Kit(D814Y) surrounded cathepsin D and dextran. (f) For visualizing recycling endosomes, RCM cells were incubated for 1 h with 5 μg ml⁻¹ AF647-cholera toxin subunit B (AF647-CTXB) and stained with anti-Kit (green). The toxin was visualized with AF647 fluorescence (blue). Magnified images of the boxed area are shown. Bar, 5 μm. (g) Pearson’s coefficients (Pearson’s R) were calculated by intensity correlation analysis of Kit(D814Y) versus organelle markers. Results are means±s.d. (n=10~20). Data were subjected to one-way ANOVA with Dunnett’s multiple comparison post-hoc test. ***P<0.001.