Fig. 5. EphB1/Fc stimulates migration of CHO cells transfected with ephrin-B1. (A) EphB1/Fc stimulates tyrosine phosphorylation of transfected ephrin-B1. CHO cells were transfected with plasmid expressing full-length ephrin-B1 as described in the Materials and Methods. 48 hours after transfection, cells were stimulated with the control IgG or EphB1/Fc (2 µg/ml), and ephrin-B1 was immunoprecipitated as in Fig. 1. Ephrin-B1 tyrosine phosphorylation (upper panel) was assessed by 4G10 immunoblot and recovery by anti-ephrin-B1 immunoblot (lower panel). (B) 48 hours after transfection with the plasmids indicated, wound closure assays were performed as described above. The migration rate is expressed as the percentage of closure/hour. The medium was replaced with serum-free medium (vehicle) or EphB1/Fc constructs as indicated in the bottom panel. Surface expression of ephrin-B1 ectodomain was analyzed by FACS analysis as described in the Materials and Methods. The data represent means±s.e.m of three independent experiments.