Overexpression of kinase-dead EphA3 blocks EphB2 activation and cell retraction. Comparison of EphB2 phosphorylation in Wt EphA3 and EphA3[KM] COS7 cell lines. (A) Parental COS7 cells (COS7) and clones stably expressing Wt EphA3 (wtA3) or high levels of K653M-EphA3 (A3KM hi) stimulated for 15 min with ephrin-B1-Fc were lysed, and α-PY Sepharose (4G10) IPs were analyzed by an α-EphB2 WB. α-EphA3 and α-EphB2 blots from total cell lysate antibodies show relative receptor expression levels. (B) Parental COS7 cells and clones stably overexpressing high levels of Wt EphA3 (wt EphA3 hi) or moderate levels of EphA3[KM] were stimulated with ephrin-B1-Fc or IIIA4, and α-EphB2 and -EphA3 IPs were Western blotted for EphB2 phosphorylation and Eph receptor expression levels, as indicated. (C) Ephrin-B1–induced cell retraction is blocked by exogenous kinase-dead EphA3. COS7 cells overexpressing Wt EphA3 (top four rows) or EphA3[KM] (bottom two rows) were labeled with (noncross-linked) Alexa Fluor 594 IIIA4 to indicate EphA3 expression, stimulated with cross-linked ephrin-B1-Fc, and analyzed by time-lapse fluorescence live-cell imaging. Cell retraction is imaged in a group of three cells that are part of a larger cell cluster (top two rows) and also in a single EphA3 Wt–expressing cell demonstrating more pronounced cell contraction (middle two rows). The numbers above the images refer to time in minutes. All scale bars are in micrometers. (D) The percentage of retracting cells with detectable EphA3 expression was quantified from B and compared with levels of parental COS7 cells (mean ± SEM; n > 3). cont, control.