The association of p85 with GPIbα is independent of phosphorylation or 14-3-3ζ binding. (A) Pull-down of GPIb-IX from human platelet lysates that were either untreated or pretreated with CIP phosphatase for 1 hour at 37°C by GST alone, GST-14-3-3ζ, or GST-p85-N. Samples were resolved by SDS 5% to 20% polyacrylamide gel electrophoresis under reducing conditions, and visualized by Western blotting with either antiglycocalicin antibody (top panel) or anti–phospho-Ser609 GPIbα antibody (bottom panel). (B) Pull-down of GPIb-IX from human platelet lysates without or with the addition of 14-3-3ζ (20 μg/mL, final concentration) by GST alone, GST-14-3-3ζ, or GST-p85-N. Samples were resolved by SDS 5% to 20% polyacrylamide gel electrophoresis under reducing conditions, and visualized by Western blotting with antiglycocalicin antibody. The results are representative of 3 separate experiments.