Fig. 3. Wild-type MEFs express Eph receptors and respond repulsively to substrate-bound ephrin stripes. Wild-type MEFs were stimulated for 0, 5, 15 and 30 minutes with 1 µg/ml pre-clustered ephrin-A5-Fc and then lysed in immunoprecipitation buffer. Endogenous EphA4 was immunoprecipitated using mouse anti-EphA4. (A) Samples were western blotted for phospho-tyrosine (p-Y) and EphA4 to demonstrate EphA4 activation and loading, respectively. Bars, 130 µm. (B-D) 6x10³ wild-type MEFs (green; F-actin staining) were plated to human Fc (negative control; B), ephrin-A5-Fc (C), or ephrin-B2-Fc (D) stripes and incubated for 18 hours. (E) Graph shows the average percentage of cells (+ 1 s.e.m. error bars, n=4) avoiding ephrin stripes and the equivalent stripe in the matched negative controls for those experiments. The difference between these values represents the repulsive response to a particular ephrin ligand (black arrow).