Fig. 4. Analysis of clathrin-dependent endocytosis. The clathrin-sequestering protein AP180C-myc was overexpressed in Oli-neu cells to interfere with clathrin-mediated endocytosis. AP180C-expressing cells were identified by staining with anti-myc antibodies (top row). Color images (bottom row) depict endocytosis (red) and cell surface localization (green/yellow). The graphs show the percentage of endocytosing AP180C-negative cells (black bars, control) and the percentage of endocytosing AP180C-expressing cells (gray bars). (A) The clathrin-mediated uptake of transferrin Alexa Fluor 594 (red) is blocked specifically in AP180C-overexpressing Oli-neu cells (arrowheads). Blue color depicts DAPI-stained nuclei. (B) Coexpression of AP180C-myc and PLP, MAG or MOG. Transfected cells were subjected to antibody-uptake experiments as described in Fig. 2B. Stacked confocal images of representative cells are shown. Overexpression of AP180C strongly reduced the internalization of MAG and MOG. More than 50 cells were counted per three (PLP and MAG) or four (MOG) independent experiments. Error bars represent s.e.m.; ^**P<0.01; ^***P<0.001 (paired t-test). Scale bars: 5 µm.