P-selectin–triggered signaling through PSGL-1 does not requireL-selectin. (A) Flow cytometric analysis of expression of L-selectin, PSGL-1, CD44, or β2 integrins on WT or Sell^−/− neutrophils. (B) Rolling velocities of WT or Sell^−/− neutrophils on P-selectin with or without coimmobilized ICAM-1 in the presence or absence of anti–ICAM-1 mAb. (C) Rolling velocities of WT or Sell^−/− neutrophils on P-selectin coimmobilized with ICAM-1 and low-dose CXCL1 (0.1 μg/ml) in the presence or absence of anti–ICAM-1 mAb. (D) Percentages of WT or Sell^−/− neutrophils rolling, arrested and round, or arrested and spread on coimmobilized P-selectin, ICAM-1, and low-dose CXCL1. (E-H) Bone marrow leukocytes from WT or Sell^−/− mice were incubated on immobilized control CD45-IgM or P-selectin–IgM in the presence or absence of EDTA. Lysates were analyzed by immunoblotting with the indicated antibodies. (I) Isolated bone marrow neutrophils from WT mice were pretreated with DMSO (vehicle control), inactive control α-cyclodextrin (αCD), or methyl-β-cyclodextrin (MβCD). They were then incubated on immobilized F(ab′)₂ fragments of anti–PSGL-1 mAb or anti–L-selectin mAb. Lysates were analyzed by immunoblotting with antibody against SFK or phospho-SFK. The data in panels B through D represent the mean ± SEM from 5 experiments, with 5 mice in each experimental group. The data in panels A and E through I are representative of 3 experiments. *P < .05.