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Fig. 5. Analysis of clathrin-independent endocytosis. To inhibit clathrin-independent cholesterol-dependent endocytosis, Oli-neu cells transiently expressing PLP, MAG and MOG were treated with the cholesterol-depleting agent filipin (1 µg/ml) during antibody uptake. To exclude that cholesterol depletion affects clathrin-dependent endocytosis, transferrin-FITC (green) was co-endocytosed with the myelin proteins and cells internalizing transferrin were exclusively analyzed. Cell-surface PLP, MAG or MOG was counterstained in blue. In merged images, surface-localized proteins thus appear magenta, whereas endocytosed proteins appear red (or yellow in case of colocalization with transferrin-FITC). Confocal images of representative cells are shown. The percentage of cells internalizing PLP, MAG or MOG was calculated from the population of transferrin-internalizing cells (graphs: black bars, untreated cells; grey bars, filipin-treated cells). More than 50 cells were counted per three (PLP and MAG) or five (MOG) independent experiments. PLP endocytosis was inhibited by filipin treatment, whereas endocytosis of MAG and MOG was unaffected. Error bars represent s.e.m.; **P<0.01 (paired t-test). Scale bars: 5 µm.
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