S655 phosphorylation-dependent APP retrieval occurs via VPS35-containing endocytic vesicles. (A) APP-GFP transfected COS-7 cells were subjected to the 15 min 22C11 uptake assay (Alexa350 blue staining) and the co-localization of endocytosed APP-GFP proteins (blue/green vesicles) with the VPS35 retromer subunit (Texas red staining) was monitored. The Golgi area denotes the highest degree of co-localization, and a region was magnified for better visualization of the tubulating vesicles (ROI-4.0-fold magnified; arrows point to APP-GFP/AU22C11/VPS35-positive structures for Wt and S655E, and arrowhead to a S655A APP-GFP-negative, AU22C11/VPS35-positive structure). Some of the tubulating vesicles for Wt and S655E have been represented schematically with a black line. (B) Co-localization between APP-GFP or 22C11 uptake with VPS35 was quantified using confocal software. Values are mean ± SEM, n = 30 cells. Statistical significance symbols: (*), S655 phosphomutant vs Wt data; (+), S655A vs S655E data. Statistically significant levels are presented as (**) for p < 0.01; and (***/+++) for p < 0.001. (C) Secreted Abeta peptides from 3 h conditioned media of APP-GFP or EGFP vector ("V") transfected COS-7 cells. Samples were immunoprecipitated, separated on urea gels and immunostained with mAb 1E8. Upper panel: Immunoblot analysis of cells lysates using the anti-APP antibody 22C11. Bands a and b, transfected immature and mature APP-GFP forms, respectively. The various Abeta species were quantified, corrected for the relative respective APP-GFP expression levels (bands a+b), and plotted as fold-increase of total (sum of all species) Abeta fragments over Wt values (mean ± SEM, n = 3).