U18666A and bafilomycin induce accumulation of KRas in a Rab7/LBPA late endosomal compartment. (A) HA-KRas and GFP-Rab7–expressing cells were treated with EGF for 20 min and immunolabeled with an anti-HA (red channel) and anti-EEA1 (blue channel). Arrows indicate the colocalization of KRas and EEA1, and arrowheads point to Rab7-positive structures. (B) Localization of GFP-KRas in cells treated with U18666A. Arrows point to PM KRas labeling, and arrowheads show intracellular ringlike structures. (C) HA-KRas was coexpressed with GFP-Rab7 and treated with U18666A plus EGF for 45 min. Arrows show colocalization of HA-KRas with GFP-Rab7. (D–F) Localization of KRas in cells treated with 20 nM B-A1 overnight and with 100 ng/ml EGF for 45 min. Arrows indicate colocalization of HA-KRas and Rab7 (E) or GFP-KRas and LBPA (F) on enlarged endosomes. The white dotted contour in D indicates the cell perimeter, and the arrowheads point to ringlike endosomal structures. Bars: (A–D) 8 µm; (E and F) 2 µm.