Figure 6: Kit(D814Y) at the ER activates STAT5 in mouse cells.

(a,b) Inhibition of export of Kit(D814Y) from the Golgi. RCM cells were cultured with 250 nM monensin (inhibits Golgi transport) for 24 h. (a) RCM cells were stained with anti-Kit (green) and anti-GM130 (Golgi marker, blue). Magnified images of the boxed area are shown. Bars, 10 μm. The graph shows correlation coefficient (Pearson’s R) between Kit and GM130. Results are means±s.d. from 15 cells. ***P<0.001, Student’s t-test. (b) Immunoblots of anti-Kit immuoprecipitates (left) and cell lysates (right). (c–e) Inhibition of export of Kit(D814Y) from the ER. RCM cells were cultured with 1 μg ml⁻¹ tunicamycin for 24 h, or 5 μM BFA for 16 h to block ER export. (c) RCM cells were stained with anti-Kit (green) and anti-calnexin (ER marker; red). Magnified images of the boxed area are shown. Bars, 10 μm. The graph shows the correlation coefficient (Pearson’s R) between Kit and calnexin. Results are means±s.d. from 11 to 13 cells. Data were subjected to one-way ANOVA with Dunnett’s multiple comparison post-hoc test. ***P<0.001. (d) Immunoblots of anti-Kit immuoprecipitates and cell lysates. NB: ER-localized Kit(D814Y) activated STAT5. (e) BFA-treated cells were stained with anti-Kit (green) and anti-STAT5 (red) antibodies. Magnified images of the boxed area are shown. Bars, 10 μm. The graph shows the correlation coefficient (Pearson’s R) between Kit and STAT5 in the non-nuclear region. Results are means±s.d. from 16 cells. ***P<0.001, Student’s t-test. NB: accumulation of Kit(D814Y) in the ER significantly enhanced co-localization with STAT5. (f) RCM cells were treated with 5 μM BFA, 1 μg ml⁻¹ tunicamycin, 250 nM monensin or 100 nM BafA1 for the indicated periods and then immunoblotted. (g) Starved R cells (left) or pt18 cells (right) stimulated with 50 ng ml⁻¹ SCF for 5 min then lysed and immunoblotted. NB: normal Kit was unable to activate STAT5. (h–j) RCM cells or starved R cells treated with 250 nM monensin or 5 μM BFA for the indicated periods, then immunoblotted. CG=complex-glycosylated form; DG=deglycosylated form; HM=high mannose form.