Fig. 5. Dose-dependent inhibition of cell adhesion and spreading by surface-bound ephrin-A5. (A) Capacity of Protein A-coated 96-well plates for Ephrin-A5 Fc () and of Protein A-bound ephrin-A5 for EphA3 (). Ephrin-A5 Fc was applied at indicated concentrations and bound protein in the supernatant was estimated by Biacore analysis. To confirm the competence of tethered ephrin-A5 to interact with the receptor, the nonbound fraction of EphA3 (2 µg/ml) after incubation on ephrin-A5-coated wells was determined by Biacore analysis. The amount of bound EphA3 estimated from this assay is shown (). (B) 293T cells, transiently transfected with w/t or mutant EphA3, and pEGFP-actin were plated (5x10⁴cells/well) onto wells coated with ephrinA5-Fc at the indicated densities (ng/mm²). After 5 hours, adherent cells were fixed with 4% paraformaldehyde and examined by fluorescence microscopy. Sections of w/t EphA3-transfected cells are shown. Bar, 20 µm. (C) Following microscopy, adherent cells were quantified using crystal violet staining. Cell attachment is expressed as a percentage (mean±s.d. from three independent assays) relative to adhesion seen on non-ephrin-A5-coated wells; [UNK], w/t EphA3; , Y596F EphA3; , Y602F EphA3; , Y779F EphA3; , Y596F+Y602F EphA3.