Endocytosed NgCAM traverses the NEEP21-positive EE. (A) Neurons were cotransfected with NgCAM and either dominant-negative Ti-VAMP (left) or anti-sense NEEP21 (right). GFP was expressed as a control. 18 h after transfection, surface NgCAM was detected with immunostaining. Coexpression of AS-NEEP21 but not Ti-VAMP-DN led to a significant decrease in the A/D PI (see Materials and methods). Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. n = 4 independent experiments, scoring 20–25 cells per experiment for each condition. (B) NgCAM-expressing neurons were allowed to endocytose anti-NgCAM antibodies for 20 min before fixation. Endocytosed NgCAM was detected with a red secondary antibody, whereas endogenous NEEP21 was detected with a rabbit anti-NEEP21 antibody (green). Precise overlap of NgCAM and NEEP21 is observed (colocalization appears yellow). Single channels as well as overlaid channels are shown for the boxed area. A single confocal section is shown. Arrowheads indicate dendrites. (C) Extent of overlap was scored for cells loaded with anti-NgCAM antibodies for 20 min without a chase (left; t = 0) and after a 1-h chase (right; t = 60). Extent of overlap was binned into strong colocalization, NEEP only, and NgCAM only. NgCAM showed high colocalization (black) at t = 0, which diminished with time (right), whereas the no colocalization categories NEEP only and NgCAM only increased with time. (D) The EE populations enriched in EEA1 (red) or in NEEP21 (green) show poor colocalization. Single channel panels are shown on the left.