Fig. 5. Heparanase releases an FGF10-FGFR2b complex bound to ECM in a solid-phase assay, and pretreatment of the ECM by heparanase decreases FGF10-FGFR2b but not FGF1-FGFR2b binding. (A) FGF10-FGFR2b or FGFR2b alone, was incubated in a 96-well plate precoated with a laminin-111 ECM that contains 2% perlecan by ELISA (data not shown). Either inactive (control) or active heparanase was added to release the bound complex, and FGFR2b was detected by ELISA. Pretreatment of the ECM with heparanase resulted in a larger decrease in binding of both the complex and the receptor alone. (B) The binding of FGF10-FGFR2b was decreased by both heparanase and heparitinase treatment; however, FGF1-FGFR2b could still bind the remaining HS after heparanase but not heparitinase treatment. ECM-coated wells were incubated with or without inactive heparanase (control), active heparanase, bacterial heparitinase, or chondroitinase for 1 hour, followed by incubation with FGF10-FGFR2b or FGF1-FGFR2b (1 nM each) for 1 hour. ELISA assays were performed in triplicate and repeated at least three times.