Identification of protein domains responsible for GPIbα/FLNa interactions. (A) Amino acid sequence of the synthetic 22-mer peptide (GPIbα556-577) that binds FLNa and was used for crystallography. (B) Binding of purified recombinant human FLNa (2μg 400μL^-1) to GST-GPIbα515-610 (5μg 400μL^-1) is inhibited by the 22-mer peptide, GPIbα556-577, in a dose-dependent manner. (C) Full-length FLNa (2μg 400μL^-1) binds the GPIbα556-577 peptide immobilized on Sepharose 4B but not Sepharose 4B alone. (D) In vitro binding of GST-IgFLNa17, -18, and -19 (2 μg each) to the GPIbα556-577 peptide beads. Top and bottom rows indicate bound and input proteins to GPIbα556-577, respectively. (E) Tag-free IgFLNa17 binds GPIbα556-577 peptide with higher affinity than IgFLNa19. Increasing amount of purified IgFLNa17 or -19 were incubated with GPIbα556-577 peptide beads. (F) IgFLNa17 is the major binding site for GPIbα556-577 peptide. Various amounts of IgFLNa17 and 19 at 1:1 molar ratio were incubated with GPIbα556-577 peptide beads in the pull-down experiment shown. (G) FLNa17 but not FLNa19 interferes the binding of full-length FLNa to GPIbα peptide beads. Various amounts of FLNa17 or -19 were mixed with 1 μg purified full-length FLNa and then incubated with GPIbα556-577 peptide beads. (B-G) Bound proteins were resolved by SDS-PAGE, and stained with Coomassie blue.