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Fig. 1. Ephrin stimulation induces retraction and rounding of COS cells. (A) Still images from time-lapse movies of COS cells stimulated with ephrin-A1 Fc or ephrin-B1 Fc. (B) Photographs of cells stimulated with ephrin-A1 Fc or ephrin-B1 Fc for the indicated times (in minutes), or with Fc for 45 minutes as a control, and stained with rhodamine-phalloidin to label filamentous actin. (C) Endogenous EphA2 and EphB2 receptors become tyrosine-phosphorylated in COS cells stimulated with ephrin-A1 Fc or ephrin-B1 Fc, respectively. Immunoprecipitated EphA2 and EphB2 were probed with anti-phosphotyrosine antibodies and reprobed with anti-EphA2 or anti-EphB2 antibodies. (D) The YSA peptide causes EphA2 tyrosinephosphorylation. COS cells were left untreated or stimulated with the YSA EphA2-activating peptide, a control (ctrl) peptide, or ephrin-A1 Fc. Lysates were used for immunoprecipitation with monoclonal anti-EphA2 antibodies. The immunoprecipitates were probed with anti-phosphotyrosine antibodies and reprobed with polyclonal anti-EphA2 antibodies. (E) Activation of EphA2 induces COS cell retraction. COS cells stimulated as in D were stained with rhodamine-phalloidin. (F) Histogram showing the percentage of cells that have spikes at the periphery, indicating cell retraction, in a representative experiment as in D and E. Bars, 20 µm.
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