GPIb-IX-14-3-3ζ association in platelets and in GPIb-IX CHO cells with intracellular GPIbα deletions. Platelets (A) or CHO cells (B-C) were incubated with membrane-permeable EZ-link-NHS-Biotin and lysed in 1% Triton X-100. GPIb-IX complexes were immunoprecipitated with an anti-GPIb monoclonal antibody, separated by reduced 4% to 15% SDS-PAGE, and transferred to PVDF membranes. Coimmunoprecipitated 14-3-3ζ was detected using the anti-14-3-3ζ-specific polyclonal antibody C-16 and revealed by ECL. Membranes were then stripped and reprobed with streptavidin-HRP to reveal biotinylated GPIbα. The upper panels correspond to the region of the gel containing the approximately 29 kDa 14-3-3ζ band, and the lower panels correspond to the region containing the approximately 135 kDa GPIbα band. (A) In human platelets, 14-3-3ζ was coimmunoprecipitated with the GPIb-IX complex by an antibody against GPIbα or GPIbβ (for simplicity only the GPIbα band is shown). (B) In transfected CHO cells, 14-3-3ζ was coprecipitated with the wild-type (WT) GPIb-IX complex and with a complex containing a central (Δ535-569) deletion. Complete deletion of the intracellular domain of GPIbα (Δ518-610) or progressive truncations from the C-terminal end (Δ605-610, Δ595-610, Δ576-610, and Δ569-610) prevented GPIb-IX-14-3-3ζ association. (C) In cells containing a series of shorter deletions of the GPIbα 535-590 region, GPIb-IX-14-3-3ζ interaction was absent only for the Δ580-590 mutant receptor. Results are representative of 4 separate experiments.