Up-regulation of PAT1 in primary neurons results in a strong colocalization with APP in the cytoplasm but not at the cell surface. A-B. PAT1-myc was overexpressed by transfection in primary neurons at 5 DIV. 24 h later cells were fixed by PFA 4% only (A) or followed by 0.2% Triton X 100 (B). Immunolabeling for PAT1-myc (Alexa-488) and endogenous APP (Cy3) was performed and analyzed by confocal microscopy. Two representative images in each condition are presented. In (A) immunolabeling was performed using the anti-myc tag polyclonal #06-549 and the anti-APP-Nter A4 antibody. In (B) the anti-myc tag MABE282 and the anti-APP-Cter polyclonal antibodies were used for immunolabeling. Quantification of colocalization using Volocity software was expressed by Pearson’s coefficient (upper panel). Data presented are the mean ± SEM of 3 independent experiments. Quantification of total APP in conditions of 0.2% Triton X 100 was performed in 23 transfected and 42 non-transfected cells (Ctrl) out of 3 independent experiments. Data are expressed in integrated density / cell in arbitrary units (AU) and represents the mean ± SEM (lower panel). Scale bar: 10 μm. C) Cell surface biotinylation was performed on PAT1-myc overexpressed cells comparatively to control cells (Ctrl). Experiments were performed on 10.106 cells 24 h after transfection. NCAM was used as internal control of membrane loading for cell surface biotinylation. The level of APP in biotinylated membranes was reported to NCAM and expressed in arbitrary units (AU). Data are the mean ± SEM of 3 independent experiments. Western blotting were performed using the anti-APP-Nter A4 and the anti-NCAM antibodies.