A portion of E-selectin distributes in detergent-resistant fractions from HUVECs but not from transfected CHO cells. IL-1β–stimulated HUVECs or transfected CHO cells expressing wild-type E-selectin were lysed in cold 1% Triton X-100, and the lysate was centrifuged in a discontinuous Optiprep gradient. Fractions collected from top to bottom were resolved by SDS-PAGE and analyzed by Western blotting with mAbs to the indicated proteins, except for GM1, which was identified with cholera toxin B. (A,B) Representative Western blots of gradients from CHO cells and HUVECs. (C) The percentage of E-selectin in each fraction was quantified by the density of blotted signals using Adobe Photoshop software. The data represent the mean plus or minus SEM of at least 4 experiments. (D) Confocal microscopy was used to measure colocalization of E-selectin with GM1, caveolin-1, or flotillin-1 in IL-1β–stimulated HUVECs or transfected CHO cells expressing wild-type E-selectin, using methods, such as those in Figure 2 and detailed in “Methods.” The degree of colocalization was quantified by measuring the percentage of green pixels (E-selectin) that colocalized with red pixels (GM1, caveolin-1, or flotillin-1). The data represent the mean plus or minus SEM of at least of 3 experiments, with at least 10 cells counted in each experiment (*P < .05, as measured by the unpaired t test).