S655 phosphorylation is a targeting signal for retrieval of APP to the Golgi. (A) Wt, S655A and S655E APP-GFP at the cell surface were labelled (0 min) by incubating COS-7 cells with the 22C11 anti-APP ectodomain antibody ("22C11 Uptake", Texas red staining) at 4°C. Following 15 and 30 min of incubation at 37°C, the localization of APP-GFP endocytic vesicles (green/red-22C11 co-localizing vesicles) were monitored. Arrows indicate the Golgi area and APP-GFP/22C11 endocytic vesicles co-localizing to or around the same region. ROI - region of interest denoting the Golgi area and co-localizing vesicles. (B) The highly green-fluorescent perinuclear structure in APP-GFP transfected cells was confirmed to be the Golgi in Wt APP-GFP and CFP-Golgi co-transfected cells, upon 30 min of 22C11 uptake. Bar, 10 μm. (C) confocal quantitative analysis of the degree of co-localization between uptaken 22C11 and the APP-GPP population at the Golgi area at 30 min of 22C11 antibody uptake (AU) at 37°C. Values are mean ± SEM, n = 20 cells. Statistical significance symbols used were: (*) for comparison of S655 phosphomutant and Wt data; (+) S655A vs S655E data. Statistical significance levels are presented as (**), for p < 0.01; and (***/+++), for p < 0.001.