Time course of Fg and/or PAF-induced tyrosine phosphorylation of proteins in CHO-αIIbβ3-PAFR cells.
(A) Cells (2.5 × 106/mL) were incubated with 100 nM PAF for 2 minutes at room temperature in a 250 μL final volume and then Fg was added at 1 mg/mL and cells were plated onto BSA-precoated wells. (B) Cells were incubated with 1 mg/mL Fg for 15 minutes at room temperature in a 250 μL final volume and then stimulated with 100 nM PAF and plated onto BSA-precoated wells. At the indicated times, cells were lysed and proteins (50 μg) were resolved by SDS-PAGE, transferred to PVDF membranes and probed with antiphosphotyrosine mAb PY99, as described in “Materials and methods.” The arrow points to the phosphorylated MAPK protein. The densitometric analysis was carried out using the public domain program NIH Image (http://rsb.info.nih.gov/nih-image/).
