The integrin α₉β₁ is the VCAM-1-binding receptor mediating neutrophil survival. Potential VCAM-1-binding integrins expressed on neutrophils (A) or on CD4 T cells (B) were quantified using flow cytometry. (C) VCAM-1-coated beads were cross-linked to the cell surface of neutrophils, and beads were washed and run on a reducing PAGE gel as described. Control human IgG-coated beads (for bead conjugation, see “Immunoblots and Western blotting” under “Materials and methods”) were used to show nonspecific binding of surface molecules to the beads after cross-linking. Proteins were detected by Western blot with anti-α₉ and anti-β₁-specific antibodies. The blots shown are representative of 3 separate experiments performed. Neutrophils were incubated for 18 hours (D) in the presence or absence of the anti-α₉β₁ antibody Y9A2 (10 μg/mL). Cells were cultured alone (□) or in the presence of VCAM-1 (▪). An isotype-control antibody is shown. Apoptosis was measured by DiOC6 staining. Results are expressed as the percentage of apoptotic neutrophils compared with neutrophils cultured in medium alone with no VCAM-1; mean ± SD of 3 separate experiments.