Fig. 1.

PSGL-1, CD43, and CD44 require core 1-derived O-glycans to associate with lipid rafts. (A) WT or C1galt1^−/− neutrophils were lysed in cold 1% Triton X-100 and centrifuged in an OptiPrep gradient. Fractions collected from Top to Bottom (Left to Right, corresponding to lower to higher density) were analyzed by Western blotting with antibodies to the indicated proteins. (B) WT or C1galt1^−/− neutrophils were incubated with Alexa 488-conjugated CTxB (green) to label GM1-containing lipid rafts. The cells were then incubated with anti-CTxB antibodies at 4 °C as control (unpatched) or at 37 °C to aggregate the rafts (patched). The cells were then fixed and labeled with antibodies to the indicated protein, followed by Alexa 647-conjugated secondary antibody (red). Representative cells were visualized with confocal microscopy to identify CTxB, antibody (Ab), or both CTxB and Ab (merge). Results are representative of at least three experiments. (Scale bar, 5 μm.)