KRas follows the clathrin-dependent endocytic pathway upon EGF stimulation. (A–G) COS-1 cells were transiently transfected with GFP-KRas, and its intracellular localization was analyzed by confocal microscopy. (A) Starved cells were fixed and immunolabeled with an anticlathrin antibody. (B) Clathrin detection on starved cells after 1 h at 4°C. (C) Cells treated with EGF-TRITC for 2.5 min were fixed and immunolabeled with an anticlathrin antibody. The red dotted lines point to colocalization in the image and in the z-axis profile. (D) The same as in C, but cells were incubated with EGF-TRITC for 1 h at 4°C. Reconstruction of the z-axis profile was performed to confirm the visualization on basal PM (C and D, insets). (E) Cells treated with EGF-TRITC for 2.5 min were fixed and immunolabeled with anti-EGFR. (F) Cells coexpressing GFP-KRas and the HA-tagged dynamin mutant K44A (DynK44A) were treated with EGF-TRITC for 20 min and immunolabeled with anti-HA. Red dotted contours indicate the perimeter of cells cotransfected with GFP-KRas and HA-DynK44A. (G) Quantification of EEA1 and GFP-KRas colocalization in control and dynamin K44A–expressing cells ± EGF is shown. 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. (A–E) 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.