Rab25 inactivates EGFR. Cancer cells were transfected with the indicated vectors for 24 h. (A) The cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) The p-EGFR expression was visualized using immunofluorescence. Original magnification, ×400; scale bar, 20 µm. (C) Ras activity was analyzed using the Ras-GTP pull down assay. Total protein expressions were analyzed by immunoblotting with the indicated antibodies. (D) Ras activity was analyzed using the Ras activation ELISA kit (***P<0.001 vs. control vector). (E) HCT-116 cells were transfected with the indicated vectors for 24 h. Invasion was analyzed by utilizing a 48-well chemotaxis chamber (**P<0.01 vs. control vector, ***P<0.001 vs. control vector and ###P<0.001 vs. Rab25 overexpression). Representative results of at least three independent experiments with similar results. Rab25, Ras-related protein 25; p-, phosphorylated; IP, immunoprecipitation; WB, western blotting.