Endocytosed APP is present in Clathrin and Rab 5-positive vesicles tubulating vesicles. The 22C11 uptake assay (Alexa350 blue staining) was repeated in COS-7 cells for further characterization of the vesicles retrieving endocytosed APP-GFP to the TGN. Immunocytochemistry analysis using antibodies against (A) clathrin and (B) Rab 5 (Texas red staining) confirmed the presence of these proteins in APP-GFP endocytic vesicles. ROI, region of interest, 1.7- and 2.0-fold magnified, respectively. Arrows in ROIs depict APP tubulating endosomes, further magnified. While 22C11/APP-GFP and clathrin were observed to be sorted from the vacuole to the emerging tubule, Rab 5 appears to be maintained in the vacuole. Bar, 10 μm. (C) confocal quantitative analysis of the degree of co-localization between Wt APP-GPP and uptaken 22C11 with clathrin and Rab-5. Values are mean ± SEM, n = 20 cells.