Figure 2

Tyrosine phosphorylation of Pyk2 induced by fMLP in dHL-60 cells adhering to plated fibrinogen. A) dHL-60 cells were either in suspension or added to plated fibrinogen, and then stimulated with 1 μM fMLP for indicated times at 37°C. Pyk2 activation was measured by its autophosphorylation detected by Y402 phosphorylation-specific antibody (upper panel). Total Pyk2 expression was measured by Western blot using anti-Pyk2 antibody (lower panel). B) Effect of anti-β2 integrin antibody on phosphorylation of Pyk2 induced by fMLP in adherent dHL60 cells. dHL60 cells were preincubated with 10 μg/ml antibody against CD18 for 30 min, and then added to Fg-coated plates with 1 μM fMLP for 10 min. Pyk2 tyrosine phosphorylation was then measured as above. C) Effect of anti-β2 integrin antibody on phosphorylation of Pyk2 induced by fMLP in adherent dHL60 cells. dHL60 cells were preincubated with 10 μg/ml anti-CD18 antibody for 30 min, and then added to Fg-coated plates with 1 μM fMLP for 30 min. Adhesion of dHL60 cells to plated Fg was then measured as residual myeloperoxidase assay. Results are presented as the mean ± SEM from 4 separate experiments. **p < 0.01 vs. fMLP alone, Fisher's LSD test. D) Effect of TAT-Pyk2-CT on adhesion of dHL-60 cells to plated fibrinogen. dHL-60 cells were preincubated with indicated concentrations of TAT-Pyk2-CT and then adhered to plated fibrinogen for 30 min in the presence of 1 μM fMLP. Adhesion of dHL-60 cells was measured by residual myeloperoxidase activity assay. Results are presented as the mean ± SEM from 4 separate experiments.