PSGL-1 and L-selectin localization in membrane lipid rafts is required to support KG1 cell rolling on P-selectin and Jurkat cell rolling PSGL-1. (A) KG1 cell rolling on P-selectin was evaluated under a constant shear stress (1.5 dyn/cm²) immediately (▪) or 24 hours (□) after treatment (10 minutes) with various concentrations of mβCD. (B) Jurkat cell rolling on PSGL-1/μ chimera was evaluated at constant shear stress (1.5 dyn/cm²) immediately (▪) or 24 hours (□) after treatment (10 minutes) with various concentrations of mβCD. Results in panels A and B are expressed as percentages of the number of vehicle-treated rolling cells/mm² per minute (mean ± SEM, n = 4, ***P < .001). (C-F) Cell lysate fractions, separated by zonal sedimentation on discontinuous sucrose gradients, were evaluated by immunoblotting for PSGL-1 (C, E) or L-selectin (D, F). (C, E) KG1 cells. (D, F) Jurkat cells. Pattern observed immediately (C, D) or 24 hours (E, F) after cell treatment (10 minutes) with 10 mM mβCD.