Figure 5: Kit(D814Y) traffics to endolysosomes to activate Akt in mouse cells.

(a) Endolysosomes from RCM cells were immunoprecipitated with control or anti-LAMP1, then immunoblotted. (b,c) RCM cells were transfected with PH-GFP or PH(R25C)-GFP. (b) Expressed proteins were visualized by GFP fluorescence. Phase contrast images are shown. Bars, 10 μm. (c) Cells were cultured for 1 h with 1 mg ml⁻¹ AF647-dextran (a marker for endocytic compartments). Bars, 10 μm. The graph shows Pearson’s R correlation coefficient between dextran and expressed proteins. Results are means±s.d. from 19 or 21 cells. **P<0.01, Student’s t-test. (d) RCM cells were co-stained for Kit(D814Y) (green) and phosphorylated Akt (pAkt; red). Bar, 5 μm. (e–g) RCM cells were cultured with 100 nM BafA1 (blocks endosomal trafficking) or 20 mM NH₄Cl (blocks proteases) for 24 h. (e) Cells stained with anti-Kit (green) and anti-LAMP1 (endolysosome marker; red). Bars, 10 μm. The graph shows Pearson’s R correlation coefficient between Kit(D814Y) and LAMP1. Results are means±s.d. (n=10~22). Data were subjected to one-way ANOVA with Dunnett’s multiple comparison post-hoc test. ***P<0.001; NS, not significant. (f) Immunoblots of anti-Kit immunoprecipitates and cell lysates. (g) Immunoblots, RCM cells treated with 100 nM BafA1 for the indicated periods. (h) After 3-h treatment with 100 nM BafA1, R cells were stimulated with 50 ng ml⁻¹ SCF for the indicated periods in the presence of BafA1, then immunoblotted. The graph shows the percentage of pAkt after nomalization with control at 5-min stimulation. Results (%) represent means±s.d. from three independent experiments. BafA1 did not affect pAkt significantly (Student’s t-test). (i) Immunoblots, endolysosomes from R cells treated with 50 ng ml⁻¹ SCF as indicated. (j,k) R cells were stimulated with 50 ng ml⁻¹ SCF for 5 or 60 min. Expressed proteins, Kit(wt) or pAkt are shown. Bars, 10 μm. The graph shows the number of Kit(+) vesicles with pAkt. Results (%) represent means±s.d (n=8~14). NS, not significant; ***P<0.001, Student’s t-test. ND=not detected.