EphA3 rescues defective cell segregation of EphB2ΔICD cells from ephrin-B1 cells. (A) Ephrin-B1 HEK293 cells were cocultured with CellTracker green–labeled HEK293 cells expressing the indicated combinations of Wt or truncated EphB2 (ΔICD) and EphA3. At confluency (2–3 d), cells were fixed and imaged by phase-contrast and fluorescence microscopy. The scale bar shown is in micrometers. (B) Segregation of green-labeled and nonlabeled HEK293T cells was determined by measuring the tightness of (green) HEK293 cell aggregation and quantified by estimating mean fluorescence intensity per total fluorescence area (>12 random fields/condition). To illustrate equal fluorescence intensity of CellTracker green–labeled cells, insets in A illustrate comparisons of individual nonclustered cells (magnified from areas defined by dashed boxes) with those selected from the coculture of ephrin-B1/HEK293T and (green) Wt EphB2/EphA3 cells with maximal cell segregation. Statistical relevance of observed differences between all groups was determined using the analysis of variance (ANOVA) test for multiple comparisons. Means with error bars indicating 95% confidence intervals (ANOVA) are shown. (C) EphA3 is activated in response to ephrin-B1-Fc in Wt EphB2 and EphB2[ΔICD]-coexpressing cells but not in cells lacking EphB2. HEK293T cell lines illustrated in A were analyzed for EphA3 phosphorylation in response to cross-linked ephrin-B1-Fc by analyzing α-EphA3 IPs by an α-PY WB. To test ephrin-B1 stimulation of EphA3, EphA3/HEK293T cells were incubated with ephrin-B1-Fc, IIIA4 (positive control), or ephrinB1/293 cells. To control loading, lysates were blotted for EphA3 and EphB2 levels.