(A through C) Complex formation of PF-4 with FGF-2. (A) Cross-linking experiments in solution. One microgram of PF-4 and 10 ng¹²⁵I–FGF-2 were incubated in 400 μL PBS in the presence or absence of 0.8 mmol/L Ca⁺⁺ and 0.5 mmol/L Mg⁺⁺ for 1 hour. Subsequently, 1 mmol/L BS³was added for another 30 minutes. At the end of the incubation period, extraction buffer was added and aliquots were loaded onto a 12% SDS-PAGE. The dried gel was analyzed by PhosphorImager or autoradiography. (B) Binding of ¹²⁵I–PF-4 to cell surface immobilized FGF-2. Fifteen nanograms per 50 microliters of FGF-2 was adsorbed onto the surface of a 96-well ELISA plate and incubated with different concentrations of ¹²⁵I–PF-4. Binding was performed and analyzed as indicated in Materials and Methods. Scatchard plot is shown as inset. The figure depicts an representative experiment done in triplicates (data points as mean; SD values: 24 < SD < 780). (C) Competion of ¹²⁵PF-4 binding to FGF-2. Wells were coated with FGF-2 at 15 ng/50 μL. Some of the wells were preincubated with 200 ng heparin. Plates were washed twice with buffer A to remove unbound heparin. Others wells received buffer alone. Ten nanograms¹²⁵I-PF-4, with or without 5 μg PF-4, 5 μg FGF-2, or 50 ng heparin, was added to the wells. Binding was performed and analyzed as indicated in Materials and Methods. The figure depicts a representative experiment done in triplicates (data points as mean + SD). 100% corresponds to 7,000 cpm.