Soluble E-selectin induces Src and Akt activation in neutrophils. Freshly isolated neutrophils were incubated in the presence or absence of PP2 (5 μM, 15 minutes) or LY294002 (10 μM, 15 minutes), with soluble E-selectin (5 μg/mL, 15 minutes). Western blots of neutrophil lysates were carried out as described in “Materials and methods,” and probed with (A) phospho-Src (Tyr 116) antibody (1:500) or (B) antiphospho-Akt1/PKBα antibody (Ser473; 1:200). To verify equal loading, the blots were probed with β-actin (1:10 000) or total Akt (1:2000). This figure is a representative blot from 3 independent experiments that were performed with similar results.