E-selectin and P-selectin signaling in human neutrophils. (A-D) Whole human heparinized blood was treated with (A) Src family kinase inhibitor PP2 (15μM for 30 minutes at RT), (B) SYK inhibitor piceatannol (25μM at 30 minutes at RT), (C) PLC inhibitor U-73122 (10μM for 30 minutes at RT), or (D) p38 MAPK inhibitor SB203580 (10μM for 30 minutes at RT), or SB203580 (10μM) plus anti–LFA-1 antibody (10 μg/mL for 20 minutes at RT), then perfused through flow chambers coated with E-selectin with or without ICAM-1 or P-selectin with or without ICAM-1. (E) Human purified neutrophils were plated on blocked (control), E-selectin–coated, or P-selectin–coated wells, and incubated under shear (65 rpm) for 10 minutes and lysed. Lysates were immunoprecipitated with anti-FGR antibody, followed by immunoblotting with a pan-Src antibody that recognizes PY416. (F) U937 cells were transfected with a constitutively active FGR or with empty vector. The binding of KIM127 or mAb 24 was analyzed by flow cytometry. Data presented are mean ± SEM and are representative of 3 independent experiments. (G-H) Human purified neutrophils were plated on blocked (control), E-selectin–coated, or P-selectin–coated wells and incubated under shear (65 rpm) for 10 minutes and lysed. (G) Lysates were immunoprecipitated with anti-SYK antibody, followed by immunoblotting with phosphotyrosine antibody 4G10. (H) Lysates were immunoblotted with antibody to detect phosphorylated p38 MAPK (phospho p38) or total p38. Data are representative of 3 independent experiments.