Analysis of deletion mutants of GPIbα expressed in CHO cells for binding to 14-3-3ζ and p85. (A) The sequence of the cytoplasmic domain of wild-type GPIbα showing the location of the central region that binds filamin^28,29  and the C-terminal 14-3-3ζ–binding sequence^6-9  (□). The location of sequences deleted in mutant GPIbα used in pull-down experiments is indicated by the shaded bars. (B) Western blot with antiglycocalicin of lysates of CHO cells expressing wild-type GPIbα or GPIbα deletion mutants or CHOβIX cells lacking GPIbα. (C) Pull-downs of CHO cell lysates with GST alone, GST-14-3-3ζ (top panel), or GST-p85-N (bottom panel). Samples were resolved by SDS 5% to 20% polyacrylamide gel electrophoresis under reducing conditions, and visualized by Western blotting with antiglycocalicin antibody (α-glyc.) as described in “Methods.” The reason for the doublet in the Δ535 to 545 GPIbα-expressing cells was not investigated. The results are representative of 3 separate experiments. WT indicates wild-type GPIbα.