Sulf-2 activity in MCF-7 CM. (A) Endosulfatase activity of CM on heparin as a function of CM volume (left panel, 4 h incubation time) and reaction time (right panel, 10 μl of CM). (B) Binding of ligand (VEGF165, FGF-2 or FGF-1) to immobilized heparin-BSA as a function of ligand concentration with or without CM treatment of heparin-BSA. Δ denotes binding of ligand to untreated heparin-BSA and ● denotes binding of ligand to CM treated heparin-BSA in pre-binding assay. Means of triplicate determinations ± S.D. are shown. In those cases where the error bars are not apparent, the S.D. values are smaller than the symbols. (C) "Pre-binding effect" of CM on ligand binding (VEGF165, black triangles; FGF-2, black squares; FGF-1, open diamonds) as a function of CM volume. These results are representative of two independent experiments (D). "Post-binding effect" of CM on ligand binding (VEGF165, black triangles; FGF-2, black squares; FGF-1, open diamonds) as a function of CM volume. Means of triplicate determinations are shown. S.D. values for FGF-1 are indicated. The S.D. values for VEGF and FGF-2 are smaller than the symbols. (E) Immuno-depletion of HSulf-2 protein by specific antibodies. MCF-7 CM was precleared with Sulf-2 antibodies (H2.1 and H2.3) or control IgG conjugated to beads and then blotted for Sulf-2 protein (H2.3). Arrows denote specific bands detected in the untreated MCF-7 CM (~240, 135 and 72 kDa). The ~50 kDa bands indicated by the asterisk are derived from the IgG used for the precipitation (F). Immuno-depletion of HSulf-2 activity by specific antibodies. The precleared CMs in E were tested in a pre-binding assay for effects on VEGF binding to heparin-BSA. The partial depletion of activity by control IgG reflects the tendency of Sulf-2 to bind nonspecifically to Sepharose beads (not shown). Means and S.D. values are shown for triplicate determinations. Statistical analysis was carried out using Student's t test. **, p < 0.01.