Figure 1. The specific binding of OPN to heparin and HSPG, a syndecan-4. (A) Biotinylated heparin binds to the nonglycosylated form of full-length OPN. Plates were precoated with recombinant OPN, derived from E. coli, as indicated. (B) Biotinylated heparin binds to the synthetic peptide corresponding to one of two putative HBDs of OPN. (C) The binding of 3 μg/ml of recombinant Syn4Ig to the plate precoated with either the full-length or thrombin-cleaved form of human OPN (OPN or T-OPN, respectively), or 10 μg/ml of the full-length form of mouse OPN (mOPN). In some experiments, the HS or CS chain of Syn4Ig was digested by either HSase or CSase, respectively. Data are presented as means ± SEM. **, P < 0.005. (D) The interaction of the native form of OPN and syndecan-4. The ability of human cell lines to secrete OPN, syndecan-4, and HS were listed. NRC-12 or OPN/Namalwa cells were stimulated with PMA. Culture supernatants were applied to the affinity column coupled with anti–syndecan-4 antibody, anti-OPN antibody, or control antibody. After washing, eluates were applied to SDS-PAGE analysis, transferred to membranes, and immunoblotted (IB) with either anti-OPN or anti–syndecan-4 antibody. ND, not detected.
Figure 1.

The specific binding of OPN to heparin and HSPG, a syndecan-4. (A) Biotinylated heparin binds to the nonglycosylated form of full-length OPN. Plates were precoated with recombinant OPN, derived from E. coli, as indicated. (B) Biotinylated heparin binds to the synthetic peptide corresponding to one of two putative HBDs of OPN. (C) The binding of 3 μg/ml of recombinant Syn4Ig to the plate precoated with either the full-length or thrombin-cleaved form of human OPN (OPN or T-OPN, respectively), or 10 μg/ml of the full-length form of mouse OPN (mOPN). In some experiments, the HS or CS chain of Syn4Ig was digested by either HSase or CSase, respectively. Data are presented as means ± SEM. **, P < 0.005. (D) The interaction of the native form of OPN and syndecan-4. The ability of human cell lines to secrete OPN, syndecan-4, and HS were listed. NRC-12 or OPN/Namalwa cells were stimulated with PMA. Culture supernatants were applied to the affinity column coupled with anti–syndecan-4 antibody, anti-OPN antibody, or control antibody. After washing, eluates were applied to SDS-PAGE analysis, transferred to membranes, and immunoblotted (IB) with either anti-OPN or anti–syndecan-4 antibody. ND, not detected.

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