The effect of phosphorylation on the association of p85 and GPIb-IX. (A) The p85 subunit of PI3-kinase. The N-terminal half of p85 (GST-p85-N, residues 1-330) and the C-terminal half of p85 (GST-p85-C, residues 325-718) were used for the pull-down experiments in panel B. (B) Pull-down of GPIb-IX from lysates of human platelets, either nonstimulated or thrombin-stimulated (1 U/mL for 5 minutes at 22°C), by GST alone, GST-p85-N, or GST-p85-C. The bait proteins were either unphosphorylated or phosphorylated with PKA/CKII prior to performing the pull-down experiments in the presence of the phosphatase inhibitor sodium orthovanadate. Samples were resolved by SDS 5% to 20% polyacrylamide gel electrophoresis under reducing conditions, and visualized by Western blotting with antiglycocalicin antibody as described in “Methods.” The results are representative of 3 separate experiments.