S655 APP phosphomutants exhibit differential cellular catabolism. (A) Wt and S655 phosphomutants turnover rates in COS-7 cells. Upper panel: Immunoblot analysis of APP-GFP transfected cells lysates using the anti-APP antibody 22C11. Bands a and b, immature (N-glycosylated) and mature (N- and O-glycosylated) APP-GFP forms, respectively, migrating above endogenous APP forms of ~115 kD and ~109 kD (potentially immature APP751/770 and APP695, respectively). APP-GFP bands identity was further confirmed using an anti-GFP antibody (data not shown). Lower panel: plotted data of the levels of immature (left graph) and mature (right graph) APP-GFP, expressed as percentage of OD values at 0 h in CHX. Values are mean ± SEM (n = 6). Statistical significance symbols: (*), S655 phosphomutants vs. Wt; (+), S655A vs. S655E; statistical significance levels are presented as (*/+) for p ≤ 0.05; (++), for p < 0.01; and (***/+++) for p < 0.001. (B) S655A is preferentially delivered to lysosomes. The targeting of Wt and S655 phosphomutants (green vesicles) to lysosomes was analysed by co-localization with the lysosomal marker cathepsin D ("Cat D", red vesicles). Arrows indicate APP-GFP-containing lysosomes (green/red co-localizing vesicles). Bar, 10 μm. (C) Alterations in the mature S655A and S655E turnover upon inhibition of lysosomal hydrolases with 50 μM chloroquine (CQ). Left: Immunoblot analysis of transfected cells lysates using the anti-GFP antibody JL-8. Right: mature S655A and S655E (band b) levels were quantified and data plotted as percentages of OD values at 0 h in CHX. Values are mean ± SEM (n = 3); (#), statistical significance of p ≤ 0.05 for CQ plus vs control data.