Fig. 7. Confrontation experiments of overexpressing cells EphB4- (clone 4-8), ephrinB2- (clone 2-6) and ephrinB2 (2-15). Equal numbers of cells were mixed and seeded at confluent cell density. Combinations of mock-transfected PAECs with either ephrinB2 (A), EphB4 (B) or ephrinB2 (C) results in complete intermingling of the two cell populations. By contrast, combinations of ephrinB2 (D) or ephrinB2 (E) with EphB4-overexpressing cells leads to segregation of the two cell populations as demonstrated by island formation of EphB4-expressing cells (red staining in A and C: EphB4-Fc receptor body staining for ephrinB2 expression; red staining in B, D and E: ephrinB2-Fc receptor body staining for EphB4). EphrinB2- or ephrinB2-mediated segregation of EphB4⁺ cells is associated with intense tyrosine phosphorylation of EphB4 (F). Biochemical analysis of confrontation experiments of mock-transfected cells with ephrinB4-transfected cells identified a weak phospho-EphB4 band. By contrast, co-culture of either ephrinB2 or ephrinB2 cells with EphB4 cells resulted in intense EphB4 tyrosine phosphorylation (F, arrowhead). In turn, analysis of ephrinB2 expression and phosphorylation identified abundant levels of full-length ephrinB2 and the truncated ephrinB2 (G, upper right arrowhead and arrow). Yet, a phospho-ephrinB2 band was only detectable in the confrontation experiments of ephrinB2/EphB4 co-cultures and in none of the other combinations (G, lower right arrowhead).