FIGURE 6.

The EGFR does not reach LAMP-2-positive lysosomes in the absence of RAB7. A, HeLa cells were transfected with nothing (No siRNA), RAB7 siRNA, or siCON and pulse-labeled with Texas Red EGF (red) for the indicated times. Cells were fixed and processed for indirect immunofluorescence staining with antibody against LAMP-2 (lysosomes) (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Size bar, 10 μm. Images were collected with an Olympus FV1000 confocal microscope and processed using the FV1000 software. Insets are higher resolution images of the boxed region. Top, Texas Red signal; middle, CD63 signal; bottom, merged image. Inset size bar, 1 μm. Shown are single planes that are representative of three independent experiments. B, endosomes positive for both LAMP-2 and Texas Red EGF were counted and plotted as a percentage of total endosomes positive for LAMP-2 only. Data are plotted as the mean ± S.E. (n = 3). Three consecutive planes from three separate fields containing 5–15 cells were analyzed from each experiment for each condition. ^*, p < 0.05 (Student's t test).