Fig. 2. Binding of versican and aggrecan G1 domains to immobilized TSP1. (A) Self displacement of TSP1 binding to versican G1 domain. Wells coated with 0.26 µg per well of VG1 were incubated at 37°C with 0.25 µg ml^–1 [¹²⁵I]-TSP1 in the presence of unlabelled TSP1 at 0.25-100 µg ml^–1 in buffers containing Ca²⁺ and Mg²⁺ () or 5 mM EDTA (). The curves represent the best fit displacement curves calculated for a single site model using the program LIGAND. (B) Wells were coated using 0.5 µg per well TSP1, blocked with 3% BSA for 1 hour and incubated with the indicated amounts of biotinylated G1 domains of versican (B) or aggrecan (C) at 37°C for 3 hours in a final volume of 50 µl of DPBS with Ca²⁺ and Mg²⁺ containing 0.5% BSA and 0.1 mM phenylmethylsulfonyl fluoride. After washing to remove unbound proteins, the wells were incubated with 1 µg ml^–1 [¹²⁵I]-streptavidin for 1 hour at room temperature, washed and the bound radioactivity quantified. Background values were subtracted from each data point (1150.8±99.2).