OPN stimulates in vitro activation of integrin receptors to form focal adhesions at the apical surface of oTr1 cells. A) Cartoon illustrating a polystyrene bead coated with recombinant rat OPN containing an intact RGD integrin binding sequence, and allowed to settle onto a cultured oTr1 cell. Note the illustrated representation of aggregated integrins, indicative of focal adhesion assembly, at the interface between the surface of the bead and the apical membrane of the cell[52, 64, 66]. B) Immunofluorescence co-localization (left panels) to detect the aggregation of αv integrin subunit (right panels) and talin middle panels), an intracellular component of focal adhesions, around beads coated with recombinant rat OPN containing an intact RGD integrin binding sequence (RGD) or coated with recombinant OPN containing a mutated RAD sequence that does not bind integrins. Optical slice images from the apical plasma membrane of oTr1 cells are shown. Note the apical focal adhesions represented by immunofluo rescence co-localization (yellow color) of the integrin αv subunit with talin that results from integrin activation in response to binding of intact OPN on the surface of the bead. No apical focal adhesions were induced by beads coated with mutated OPN as evidenced by lack of integrin αv and talin aggregation around the bead.