Translocation of KRas to the early endosomal compartment is a CaM- and PKC-independent process. COS-1 cells expressing GFP-KRas or the indicated mutants were treated with EGF-TRITC for 20 min, fixed, and immunolabeled with anti-EEA1. (A) Colocalization of GFP-KRas (wild type [wt]), EEA1, and EGF-TRITC on intracellular vesicles was observed after treatment with 10 µg/ml of the CaM inhibitor W13 for 1 h. (B) The same as in A but using 10 µM of the general PKC inhibitor BIM for 1 h. (C) YFP-KRas (Ser181D [S181D]) colocalizes with EEA1 and EGF-TRITC. (D) Similarly, YFP-KRas (Ser181A [S181A]) colocalizes with EEA1 and EGF-TRITC. (E) The presence of YFP-KRas (Ser181A) on EEs was analyzed in cells treated with W13. Quantification of colocalization is shown underneath each panel. Statistical significances of differences between control and EGF treatment were determined using the Student's t test. Data are means ± SEM; **, P < 0.01; ***, P < 0.001. 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.