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Fig. 7. NCAM140 is re-expressed at the cell surface after endocytosis. (A) NCAM140 expressing B35 cells were biotinylated at 4°C and unreacted biotin was quenched. As positive control cells were directly fixed and stained for cell surface NCAM (a, anti-NCAM and anti-mouse Alexa-Fluor-488) and biotinylated cell surface proteins (a', streptavidin-Cy3). Biotin was removed from the cell surface using MESNA (+MESNA). (b-h and b'-h') Cell surface NCAM staining with anti-NCAM antibody and anti-mouse Alexa-Fluor-488-conjugated secondary antibodies (b-h); b'-h' shows the same images stained with Cy3-conjugated streptavidin for the detection of cell surface biotinylated proteins without or with induction of NCAM endocytosis (–E, E) and without or with additonal time for recycling (–R, R) for 30 or 60 minutes (30', 60'); b, b': without endocytosis and recycling (–E/–R); c, c', d, d': 30 or 60 minutes induction of NCAM endocytosis, no recycling time (30'E/–R or 60'E/–R, respectively); e, e', f, f': 30 minutes endocytosis and 30 or 60 minutes recycling time (30'E/30'R or 30'E/60'R, respectively); g, g', h, h': 60 minutes endocytosis and 30 or 60 minutes recycling time (60'E/30'R or 60'E/60'R, respectively). Overlays of the images are shown in insets. (B) Cortical neurons isolated from E15.5 mice (NCAM^+/–) were transiently transfected with NCAM140 cDNA. Cells were processed as described in A. In all images the cell surface biotin was removed using MESNA (+MESNA). The endocytosis was either induced for 30 or 60 minutes followed by 0, 30 or 60 minutes recycling time; a, a': 60'E/–R; b, b': 30'E/60'R; c, c': 60'E/30'R; d, d': 60'E/60'R.

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