Figure 1

APP and PAT1 colocalize poorly at the cell surface of primary neurons. A) Neurons at 5 DIV were fixed in PFA 4% for 30 min (left panel). After fixation, an additional permeabilization in 0.2% Triton X 100 was performed (right panel). In both cases cells were immunolabeled for Giantin or PAT1. Immunocytochemistry was analyzed by confocal microscopy. One representative immunocytochemical staining out of 4 independent experiments is presented. Scale bar: 10 μm. B) APP and PAT1 double immunolabeling was performed in neurons at 5 DIV fixed with PFA 4% only (left panel) and followed by 0.2% Triton X 100 (right panel). Anti-APP Cter polyclonal and monoclonal PAT1 antibodies were used as primary antibodies. Pearson’s coefficient was evaluated following confocal microscopy analyses and quantifications in Volocity software. Data presented are the mean ± SEM of 4 independent experiments. C) Ratio of APP to PAT1 before (Input) and after immunoprecipitation of APP in total extracts (Total Co-IP) and after cell surface biotinylation (Cell surface Co-IP). Immunoprecipitation of APP was performed with the anti-APP Cter polyclonal antibody. 10.10⁶ cells at 6 DIV were used for each condition. 40 μg of cell extracts before immunoprecipitation were loaded (Input). Control immunoprecipitation in absence of primary antibody is presented (Ctrl). Detection of APP in western blotting was performed using the anti-APP-Nter A4 antibody. Data presented are the mean ± SEM of 3 independent experiments. Representative immunoblot and histogram of the ratio of APP to PAT1 in arbitrary units (AU) are presented.