Sulf-2 effects on pre- and post-binding assays for VEFG and FGF-2. (A) Solid phase binding assays to measure "pre-binding effect" and "post-binding effect" are schematically shown. HSulf-2 was incubated with immobilized heparin before or after adding heparin-binding factors. Specific primary antibodies quantified the amount of factor bound to immobilized heparin. (B) Binding of ligand (VEGF165, FGF-2 or FGF-1) to immobilized heparin-BSA as a function of ligand concentration with or without Sulf-2 treatment of heparin-BSA. Δ denotes binding of protein to untreated heparin-BSA and ● denotes binding of VEGF, FGF-2 or FGF-1 to rHSulf-2 treated heparin-BSA in pre-binding assay. (C) "Pre-binding effect" of rHSulf-2 (●) or rΔCCHSulf-2 (○) on ligand binding (VEFG or FGF-2) to heparin-BSA as a function of enzyme concentration. (D) "Post-binding effect" of rHSulf-2 (●) or rΔCCHSulf-2 (○) on ligand binding (VEFG or FGF-2) to heparin-BSA as a function of enzyme concentration. All the results shown are representative of two independent experiments.