VPS35 down-regulation impairs S655E APP-GFP retrieval to the TGN and decreases its turnover rate. (A) Left: Immunodetection of VPS35 levels in COS-7 cells co-transfected with N1 EGFP and several concentrations of anti-VPS35 siRNAs for 24 h. Tubulin was probed as a control. Right: Quantification of VPS35 levels, plotted as percentages of control conditions (cells only transfected with N1 EGFP). Values are mean ± SEM (n = 2-6); (#), statistical significance of p ≤ 0.05 for siRNA VPS35 vs control data. (B) The 15 min 22C11 antibody uptake assay (Alexa350 blue staining) was applied to monitor S655E APP-GFP protein retrieval from the endosome to the TGN upon COS-7 cells pre-incubation with 5nM VPS35 siRNAs for 24 h to downregulate VPS35 (Texas red staining). (B.I) Control: cells only transfected with S655E APP-GFP. (B.II - B.III) siRNA VPS35: cells co-transfected with S655E APP-GFP and VPS35 siRNAs; in the majority of the population, the APP-GFP signal at the Golgi was not visible (B.II), while in a minor percentage of cells APP-GFP was still visible at the Golgi (B.III). Bar, 10 μm. (C) S655E APP-GFP turnover rate in CHX upon cells pre-incubation with 5nM VPS35 siRNAs for 24 h. Left: Immunoblot analysis of COS-7 transfected cells lysates using the anti-APP 22C11 and anti-tubulin antibodies. Bands a and b, immature and mature APP-GFP forms, respectively, migrating above endogenous APP forms. Right: mature S655E levels were quantified and plotted as percentages of OD values at 0 h in CHX. Values are mean ± SEM (n = 3); (#), statistical significance of p ≤ 0.05 for siRNA VPS35 plus vs control data.