The effect of cytoskeletal disruption or GPIbβ phosphorylation on the interaction of 14-3-3ζ or p85 with GPIbα. (A) Lysates of human platelets that were untreated, pretreated with PGE₁ prior to lysis, or lysed in the presence of NEM, were immunoprecipitated (I.P.) with rabbit antiglycocalicin (Anti-glyc.) or nonimmune control (Cont.) IgG and Western blotted with antifilamin monoclonal antibody; then, the same filters were reprobed with anti-GPIbα (WM23) as indicated (P.L. indicates platelet lysate). (B) Pull-down of GPIb-IX from lysates of human platelets that were untreated, pretreated with PGE₁ prior to lysis (that increases phosphorylation of GPIbβ), or lysed in the presence of NEM (that dissociates filamin from GPIbα), by GST alone, or GST-p85-N. Results are representative of 5 separate experiments. (C) Pull-down of GPIb-IX from human platelet lysates in the absence or presence of either a 14-3-3ζ inhibitor peptide (R18; 100 μM, final concentration) or a peptide based on the phosphorylated C-terminus of GPIbα, RYSGHpSL (α604-610; 100 μg), by GST alone, GST-14-3-3ζ, GST-p85-N, or GST-BCR. 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.” Results are representative of 3 separate experiments.