Fig. 4. Example of FRET reflecting the interaction between APP and BACE in the endosomes. (A-D) Example of FRET between APP-BACE in the endosomes. H4 cells were co-transfected with BACE-V5 (A,B) and APP-myc (C,D), immunostained with anti-APP antibody (labeled by Cy3) and anti-BACE antibody (labeled by FITC), both of which are directed against ectodomains. After labeling on ice, cells were incubated at 37°C for 15 minutes. Most of the APP signal was found in endosomes (confirmed by anti-EEA antibody), and co-localized with the BACE signal. Photobleaching of the Cy3 label of APP in the endosomes (D, boxed area) led to a marked increase in the BACE fluorescent signal within the photobleached area, demonstrating FRET (B, boxed area). Bar, 10 µm. (E) FRET ratio increases of Fl_D2/Fl_D1 after photobleaching between APP and BACE in the secretory pathway, cell surface and endocytic pathway are shown. FRET measurements are shown for ectodomain interactions at 0 minutes after labeling (cell surface), 15-30 minutes after labeling (endosomes) and 60 minutes after labeling (lysosomes). FRET increases on the cell surface and in the endosomes were significantly above 1.0 (P<0.0001). FRET was not detected in the lysosomes.