Fig. 1. Endocytosis of NCAM is developmentally regulated in cortical neurons. (A) Cortical neurons isolated from E15.5 mice (NCAM^+/–) transiently expressing either human NCAM140 or NCAM180 were cultured for 1, 3 or 5 days (DIV1, DIV3 or DIV5, respectively). Endocytosis of NCAM was not induced or induced by an antibody specific for human NCAM for 30 minutes. The numbers of NCAM-positive vesicles per cell in cell somata were counted from at least three independent experiments with approximately 15 cells analysed in each experiment. ***P<0.0005; *P<0.05 (comparison between vesicle numbers of internalized NCAM at different cultivation times); ⁺⁺⁺P<0.0001 (comparison between vesicle numbers with or without endocytosis induction). (B,C) Representative images of endocytosed NCAM. Cell surface associated NCAM-antibody complexes were detected with Cy3-conjugated secondary antibodies, internalized NCAM with Alexa-Fluor-488-conjugated secondary antibodies (white arrows). Images were either taken from cell somata (B) or from growth cones or neurites of cortical neurons (C) using a confocal laser scanning microscope.