FIGURE 2.

CD151 induces subcellular translocation of Rac1 and Ras to a membrane region containing β₁ integrins. A, 293 cells were transiently transfected with expression vectors encoding CFP or CFP-fused CD151. After fixation, the cells were permeabilized and probed with mouse anti-β₁ integrin Ab or rabbit Abs against pan-Ras and Rac1. Following incubation with fluorescent secondary antibodies described under “Experimental Procedures,” the cells were analyzed by confocal microscopy. Images in the far right column represent overlapped images between CFP (blue), β₁ integrin (green), and small GTPases (red). Magnified images of the membrane area are shown in insets in fluorescent images. B and C, transiently empty vector- and CD151-transfected 293 cells (B) and stable MelJuSo mock and CD151 transfectant cells (C) were lysed in buffer containing 1% Brij 97, and the subcellular fraction was separated by ultracentrifugation using a discontinuous sucrose density gradient. Twelve fractions were collected from the top of the gradient and analyzed by immunoblotting using Abs against CD151, integrin β₁, Rac1, or EWI-2. Data shown are representative of three separate experiments with similar results.