Figure 5.

Binding of α8^tβ1-AP and cells expressing α8β1 to known ligands and to purified native OPN and recombinant GST-OPN. (A and B) Solid-phase binding assays were performed with α8^tβ1-AP in the presence of 1 mM MnCl₂. (A) α8^tβ1-AP binding to entactin, VN, FN, native OPN, and GST-OPN. Plates were coated with indicated concentrations of purified proteins. The absorbance values were normalized using the absorbance value for binding to substrata coated with 10 μg/ml FN120. Results demonstrate that α8^tβ1-AP binds to OPN and GST-OPN as well as to FN and VN. (B) Effects of peptides and an inhibitory anti-β1 antibody on α8^tβ1-AP binding to native OPN and GST-OPN. Wells coated with 5 μg/ml OPN or 10 μg/ml GST-OPN were incubated with α8^tβ1-AP with additions indicated: (Cont) no addition; (RGD) 0.1 μg/ml GRGDSP peptide; (RGE) 0.1 μg/ml GRGESP peptide; (β1) 100 μg/ml anti-integrin β1 mAb HA2/11; and (Cont IgG) 100 μg/ml control IgG. The absorbance was quantified as the percentage of control binding (no addition). (C) Cell adhesion to native OPN and GST-OPN. Cell adhesion assays were carried out with KA8 and K562 cells on plates coated with indicated concentrations of OPN and GST-OPN in the presence of 1 mM MnCl₂. The absorbance was quantified as the percentage of maximum adhesion to FN coated at 5 μg/ml. Note that only KA8 cells adhered to OPN and GST-OPN. (D) Effects of peptides and anti-integrin mAbs on KA8 cell adhesion to OPN and GST-OPN. KA8 cell adhesion assays were carried out on OPN and GST-OPN (coated at 5 μg/ml) in the presence of 1 mM MnCl₂ with additions as indicated: (Cont) no addition; (RGD) 10 μg/ml GRGDSP peptide; (RGE) 10 μg/ml GRGESP peptide; (β1) anti-β1 mAb AIIB2; (α5) anti-α5 mAb B1E5; (αvβ5) anti-αvβ5 mAb P1F6; and (Cont IgG) 100 μg/ml control IgG. Adhesion with no addition (Cont) was set at 100%. Note that KA8 cell adhesion to OPN and GST-OPN was RGD sensitive and was blocked by the anti-β1 mAb. Data represent mean ± SD of triplicate wells.