Figure 2

Down-regulation of PAT1 increases APP at the cell surface of primary neurons. Neurons at 2 DIV were treated with either PAT1 siRNAs or GAPDH siRNAs comparatively to control cells (Ctrl) in absence of treatment. A-B: After 66 h the cells were fixed and processed for PAT1 immunocytodetection (A) or extracted for western blots (B). (A) Immunocytochemistry was analyzed by confocal microscopy and quantified by Image J. Data are expressed in integrated density/cell in arbitrary units (AU). Two representative immunolabelings of PAT1 of each condition are presented. (B) 40 μg of cell extracts were loaded for western blotting. The level of PAT1 or of APP reported to actin was expressed in arbitrary units (AU). Data in A and B are the mean ± SEM of 4 independent experiments and of 3 experiments for right panel in B. C-D: After 66 h of PAT1 siRNAs or GAPDH siRNAs, the cells were fixed by and processed for APP immunodetection (C) or for cell surface biotinylation (D). (C) APP immunocytochemistry was analyzed by confocal microscopy and quantified with Image J. Data are expressed in integrated density/cell in arbitray units (AU). Three representative images of each condition are presented. (D) Cell surface biotinylation was performed on 10⁶ cells. 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. In the whole figure immunocytochemistry was performed using the anti-APP-Nter A4 antibody. The anti-APP Cter polyclonal antibody was used for western blotting in 2B (right panel) and the anti-APP-Nter A4 antibody was used in 2D. Scale bar: 10 μm.