Time course of PAF-induced tyrosine phosphorylation of FAK in CHO-αIIbβ3-PAFR cells in the presence of either soluble or solid-phase Fg.

In aggregation assays, cells were incubated with 1 mg/mL Fg for 15 minutes at room temperature and then were stimulated with 100 nM PAF and plated onto BSA-precoated wells. At the indicated times, cells were lysed and immunoprecipitation of FAK was performed using a polyclonal anti-FAK antibody. Immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine mAb PY99, as described in “Materials and methods.” In adhesion experiments, cells were added to Fg-coated wells and allowed to adhere for 15 minutes. Then 100 nM PAF was added and incubation continued until the indicate times. Cells were lysed and tyrosine phosphorylation of FAK was determined as above. The densitometric analysis was carried out using the public domain program NIH Image (http://rsb.info.nih.gov/nih-image/).