Cell-surface E-selectin colocalizes with α-adaptin, a component of clathrin-coated pits but not with caveolin-1, a component of caveolae. IL-1β-stimulated HUVECs or transfected CHO cells expressing wild-type or tail-less E-selectin were incubated in isotonic or hypertonic medium for 15 minutes, then fixed and incubated with biotinylated polyclonal antibodies to E-selectin, followed by streptavidin conjugated to Alexa 488. After permeabilization, the cells were incubated with a mAb to α-adaptin or caveolin-1, followed by donkey antimouse Ig conjugated to Cy-3. Using a confocal microscope, an optical section at several levels of each cell was examined for staining of E-selectin (green) or α-adaptin or caveolin-1 (red). Representative images of transfected CHO cells expressing wild-type E-selectin (A) or of IL-1β-stimulated HUVECs (B) revealed partial colocalization of E-selectin with α-adaptin in isotonic medium but significantly less colocalization in hypertonic medium. Bar represents 20 μm. (C,D) The degree of colocalization was quantified by measuring the percentage of green pixels (E-selectin) that colocalized with red pixels (α-adaptin or caveolin-1). The data represent the mean plus or minus SEM of at least 4 experiments, with at least 10 cells counted in each experiment (*P < .05; ***P < .001, as measured by the unpaired t test).