KRas localizes on LEs containing the p14–MP1 scaffolding complex. (A and B) COS-1 cells transiently coexpressing GFP-KRas and Xpress-tagged p14. (A) Colocalization of KRas (green channel) with p14 (blue channel) and endocytosed EGF-TRITC (100 ng/ml for 45 min) on perinuclear endosomes (insets). (B) Late endosomal colocalization of GFP-KRas and p14 after B-A1 treatment (20 nM for 6 h) in nonstimulated cells (insets). TO-PRO 3 is a nuclear marker (blue channel). (C and D) COS-1 cells were cotransfected with GFP-KRas and myc-tagged MP1. (C) Colocalization of KRas (green channel) with MP1 (blue channel) and endocytosed EGF-TRITC (red channel) on LEs (insets). (D) Triple labeling of GFP-KRas, MP1 (red channel), and cathepsin D (CatD; blue channel) is shown in cells treated with leupeptin for 8 h and EGF (100 ng/ml for 45 min; insets). Colocalization is indicated with white (merge) or red (separate channels) arrows. Dotted boxes define the areas from which the corresponding insets were generated. Bars, 20 µm.