Figure 3: Kit(D814Y) traffics from the plasma membrane to endolysosomes through its kinase activity and clathrin-mediated endocytosis.

(a) RCM cells cultured in the presence of 1 μM PKC412 (Kit inhibitor) for 24 h and stained with anti-Kit (green) and anti-calnexin (ER marker; red). Bars, 10 μm. The graph shows the percentage of cells with predominant PM localization of Kit(D814Y). Results (%) represent means±s.d. from three independent experiments (n>200 cells). ***P<0.001, Student’s t-test. (b,c) RCM cells were cultured in the presence of (b) 20 mM NH₄Cl (inhibits lysosomal proteases) for 24 h or (c) 200 μg ml⁻¹ cycloheximide (inhibits protein synthesis) for the indicated periods, then immunoblotted. Total protein levels were confirmed by Coomassie staining. (d–f) Inhibition of clathrin-mediated endocytosis by sucrose or pitstop2. RCM cells were treated with (d,e) 0.45 M sucrose or (f) 50 μM pitstop2. After 3 h, cells were stained with antibody. Bars, 10 μm. (f) Immunoblots. (g,h) Inhibition of non-clathrin endocytosis by filipin. RCM cells treated with 1 μg ml⁻¹ filipin and/or 50 μM pitstop2 were cultured for 3 h. (g) Lysates were immunoblotted. Total protein levels were confirmed by Coomassie staining. (h) Cells were stained with anti-Kit (cyan) and anti-TfR (red). Bars, 5 μm. (i,j) Ubiquitination of Kit(D814Y). Starved pt18, R and RCM cells were treated with 50 ng ml⁻¹ SCF (i) or 0.45 M sucrose (j) for 5 min. Anti-Kit immunoprecipitates were immunoblotted. Ub: ubiquitin. (k) RCM or SCF-treated R cells were cultured in 200 μg ml⁻¹ cycloheximide (CHX) for the indicated periods, then immunoblotted. The graph shows the percentage of mature Kit remaining after CHX treatment. Results (%) represent means±s.d. from three independent experiments. *P<0.05; **P<0.01, Student’s t-test. (l,m) R cells or RCM cells were transfected with Tsg101 siRNAs (Tsg1 or Tsg2) and cultured for 24 h. Lystes from R cells treated with 50 ng ml⁻¹ SCF for 30 min (l) or RCM cells (m) were immunoblotted.