Tyrosine phosphorylation of D3-adherent cells and D3-detached cells after the stimulation with soluble and solid-phase fibrinogen. (A) D3-adherent and D3-detached cells were stimulated with 1 mg/mL soluble fibrinogen for the indicated times. (B) D3-detached cells were incubated on the fibrinogen plate for the indicated times (0, 15, 30, and 45 minutes). In (A) and (B), the cell lysates were immunoprecipitated with antiphosphotyrosine MoAb (PY20) and immunoblotting analysis was performed with antiphosphotyrosine MoAb (4G10) as described in the Materials and Methods. The positions of the molecular markers are shown to the left in kilodaltons. Solid arrows and a broken arrow indicate the positions of the tyrosine-phosphorylated protein bands and the tyrosine-dephosphorylated protein band, respectively. In (C) and (D), D3-adherent and D3-detached cells were stimulated with 1 mg/mL soluble fibrinogen for the indicated times. The cell lysates were immunoprecipitated with (C) anti-FAK polyclonal antibody or (D) anti-Syk MoAb, and immunoblotting analysis was performed with antiphosphotyrosine MoAb (4G10). The blots were stripped and reprobed with the indicated antibodies. (E) The effects of disintegrin peptides on tyrosine phosphorylation of Syk were examined. D3-adherent cells were treated with 5 mmol/L RGDS, 5 mmol/L RGES, or 40 μmol/L cyclic-KGD peptide for 15 minutes at 4°C and stimulated with 1 mg/mL soluble fibrinogen for 5 minutes, and then tyrosine phosphorylation of Syk was examined as described above. (F) The effect of cytochalasin D on tyrosine phosphorylation of Syk was examined. D3-adherent cells were pretreated with 1 or 5 μmol/L cytochalasin D for 15 minutes at 37°C and then stimulated with 1 mg/mL soluble fibrinogen for 5 minutes, and tyrosine phosphorylation of Syk was examined as described above. Results are representative of 4 experiments.