Interactions of the cytoplasmic domain of E-selectin with clathrin-coated pits of CHO cells enhance leukocyte rolling under flow. (A) Neutrophils were perfused over transfected CHO cells expressing wild-type or tailless E-selectin over a range of wall shear stresses. The number of rolling neutrophils was measured after 4 minutes. The data represent the mean plus or minus SEM of at least 4 experiments. (B) Neutrophils were perfused over CHO cells expressing wild-type or tailless E-selectin in the absence or presence of 10 μg/mL anti-E-selectin mAb ES1 or anti-β2 integrin mAb IB4 at a wall shear stress of 1 dyne/cm². The number of rolling neutrophils was measured after 4 minutes. (C) Neutrophils were perfused for 5 minutes over CHO cells expressing wild-type or tailless E-selectin at a wall shear stress of 0.25 dyne/cm². Without stopping flow, cell-free buffer was perfused at the same wall shear stress for 30 seconds, and the number of cells still rolling was taken as 100%. The wall shear stress was then increased incrementally every 30 seconds, and the percentage of neutrophils still rolling was determined. The data represent the mean plus or minus SEM of 5 experiments. (D,E) The mean velocity and the variance of velocity of neutrophils rolling on CHO cells expressing wild-type or tailless E-selectin were measured. The wall shear stress was 1 dyne/cm². The data represent the mean plus or minus SEM for at least 4 experiments. (F) Fixed neutrophils were perfused over CHO cells expressing wild-type or tailless E-selectin in isotonic or hypertonic medium at a wall shear stress of 1 dyne/cm². The number of rolling neutrophils was measured after 4 minutes. The data represent the mean plus or minus SEM of at least 3 experiments (*P < .05; **P < .01; ***P < .001, as measured by the unpaired t test).