Binding isotherm (A) and cross-linking (B) of125I-70-kD fragment to cells. (A) Increasing amounts of125I-70-kD fragment were incubated with MG63 cells in the absence (control) or presence of 200 nmol/L S1P (S1P) for 45 minutes. To achieve concentrations less than 0.5 mg/L, ligand with a specific activity of 1,000 cpm/ng was added. To achieve concentrations greater than 0.5 mg/L, unlabeled 70-kD fragment was added to125I-70-kD fragment to yield mixtures with progressively lower specific activity. Specific binding was calculated by subtraction of nonspecific binding (in the presence of 500 mg/L unlabeled fibronectin) from total binding and expressed as nanograms of 70-kD fragment bound per well. Data represent the average of duplicate values. (B) Confluent MG63 cells were incubated for 60 minutes with125I-70-kD fragment (0.5 mg/L) in medium containing unlabeled 70-kD fragment (20 mg/L; NSB), control with nothing additional (CON), 200 nmol/L LPA (LPA), 10 μmol/L nocodazole (NOC), or 100 nmol/L S1P (S1P). Cell layers were then washed and incubated for an addition of 5 minutes with factor XIIIa (10 mg/L). Cell lysates were analyzed by reducing SDS-PAGE and phosphorimaging. Top, top of the stacking gel; Int, interface of the 3% stacking and 8% separating polyacrylamide gels; and 70-kD, un–cross-linked 125I-70-kD fragment. Quantitation of bands at the top, the interface, and the position of the un–cross-linked probe yielded the following ratios of density of the three bands in treated cultures compared with control: LPA/CON: 2.0, 2.2, and 1.9; NOC/CON: 2.1, 2.5, and 2.3; and S1P/CON: 2.1, 1.9, and 1.7.
