Fig. 2. Localization of APP and BACE on the cell surface and in the endocytic pathway. APP and BACE-co-transfected H4 cells were labeled by antibodies against extracellular regions (8E5 for APP, anti-myc for BACE) as described in the Materials and Methods section, and allowed to undergo endocytosis. They were fixed and permeabilized after 0, 15, 30 or 60 minutes, and the secondary antibodies were applied to detect the localization of the primary antibodies. At time 0 minutes, only the cell surface was labeled. Localization of APP and BACE are found in nearly identical clusters on the cell surface as shown in the bottom-right panel (APP in red, BACE in green). At 15-30 minutes, most signal was found in the endosomes, showing complete co-localization of APP and BACE signals. However, at 60 minutes, APP tended to localize in lysosomes, whereas BACE did not. Bar, 20 µm.