Figure 6

Disruption of HOPS complex inhibits EGFR degradation.

(A). MCF7 cells were transfected with pSuper.GFP-scramble-shRNA or shRNA-VPS41, 48 h later, the cells were starved for overnight, then stimulated with EGF for the indicated times. The protein level of EGFR was examined by western-blot using mAb against EGFR. The results demonstrated EGFR decreased quickly in scramble knocked-down cells, but the degradation of EGFR is compromised in shRNA-VPS41 knocked-down cells. Un-cropped blots/gels are presented in Supplementary Figure s11. (B). Quantitative analysis from 3 independent experiments reveals that EGFR degradation is significantly arrrested in shRNA-VPS41 knocked-down cells. (C). Immunofluorescence microscopy showed rapid endocytic clearance of endocytosed EGF-Rhodamine in scramble knockdown cells. (D). depletion of VPS41 delayed clearance of endocytosed EGF-Rhodamine as evidenced by the accumulation of endocytosed EGF-Rhodamine in structures partially overlapping with EEA1-labeled early endosome after 60 min internalization. (E). EGF-Rhodamine accumulated vesicles were co-localized with late endosomal/lysosomal markers CD63 and Lamp1 after 60 min of endocytosis in cells with VPS41 depletion. Bar = 20 μm.