Fig. 6. ROCK inhibitor (Y-27632) attenuates the inhibitory effect of EphA1 on cell spreading and migration. (A) Activities of Rho-family GTPases in HEK293 Mock, WT-EphA1–FLAG (WT) and KD-EphA1–FLAG (KD) cells were measured by immunoblot analysis as described in the Materials and Methods. (B) Quantification of A with means ± s.d. from three independent experiments. (C) The same set of cells as shown in A were plated onto FN-coated (1 µg/ml) coverslips with immobilized ephrin-A1–Fc (1 µg/ml) in serum-reduced medium containing DMSO (vehicle; middle column) or Y-27632 (10 µM; right column) for 30 minutes. Cells were fixed and stained with rhodamine-labeled phalloidin. Scale bar: 10 µm. (D) Quantification of the spreading of the cells shown in C. (E) Modified Boyden-chamber migration analysis of the same set of cells was performed as described in the Materials and Methods. Values show means ± s.d. from nine randomly selected high-power fields.