Identification of the GPIbα-binding region of p85. (A) The p85 subunit of PI3-kinase and the regions expressed as GST-fusion proteins (■). (B) Pull-down of GPIb-IX from human platelet lysates by GST alone, or by GST-p85 fragment fusion proteins: p85-N, residues 1 to 330; SH3, residues 1 to 80 (N-terminal SH3 domain); SH3-P1, residues 1 to 112 (SH3 and PRD-1 domains); SH3-BCR, residues 1 to 302 (SH3, PRD-1, and BCR domains); BCR, residues 113 to 301 (BCR domain); BCR-P2, residues 113 to 330 (BCR and PRD-2 domains); and p85-C, residues 325 to 718. (C) The BCR domain of p85 and the regions expressed as GST-fusion proteins: BCR, residues 113 to 301 (BCR domain); S231A, residues 113 to 301 (BCR domain containing a Ser231/Ala point mutation); Δ221 to 232, BCR domain containing a deletion of a putative 14-3-3ζ–binding site; BCR-N, residues 113 to 213; and BCR-C, residues 211 to 301. (D) Pull-down of GPIb-IX from human platelet lysates (PL) by GST alone, or by the GST-BCR fragment fusion proteins shown in panel C. 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.