Protein tyrosine phosphorylation after cell adhesion on immobilized ligand. (A) Protein tyrosine phosphorylation in CHO-K1 cells stably transfected with WT or mutant (α968C/β693C) αIIb/β3 after adhering to immobilized fibrinogen or BSA (in the presence of 10 μg/mL mAb LM609) in the presence or absence of 1 mM DTT for 1 hour. Total cell lysates were blotted with antiphosphotyrosine mAb 4G10 and PY20 and reblotted with anti–β-actin (lower panel). The arrows indicate the position of 3 proteins that exhibited significantly increased phosphotyrosine signal following adhesion on fibrinogen. Positions of protein molecular size marker (in kDa) are shown on the left. (B) Tyrosine phosphorylation of focal adhesion kinase (FAK) in the transfected CHO-K1 cells. Cells were adhered on fibrinogen or BSA-coated culture dish for 1 hour followed by lysis. Lysates of adhering cells were immunoprecipitated with anti-FAK mAb and blotted with anti-FAK (pY397) (rabbit antiserum) (upper panel). The same membrane was then stripped and reblotted with anti-FAK rabbit polyclonal IgG (lower panel). (C) Quantification of tyrosine phosphorylation of FAK. The intensities of blotted phospho-FAK (Tyr397) and total FAK were quantified as described in “Immunoprecipitation and Western blotting.” The extent of FAK tyrosine phosphorylation was expressed as a percentage of phospho-FAK (pY397) signals relative to total FAK signals. Results are average of 3 independent experiments. Error bars are SD.