Fig. 2. ROCK inhibitor Y27632 antagonizes the ephrinA1-mediated repression of VSMC spreading whereas ephrinA1 does not modulate RhoA activity. (A) Representative phase-contrast microscopy of quiescent VSMCs allowed to spread on tissue culture dishes coated with laminin (0.25 µg/cm²) and 1 µg/cm² of either Fc (L+Fc) or ephrinA1/Fc (L+A1), in the presence of 7 µM Y27632 or vehicle alone. Bar, 20 µm. (B) Quiescent VSMCs were seeded on tissue culture dishes coated with laminin and either Fc or ephrinA1/Fc (A1). Cells were harvested at the indicated times and processed for pull-down experiments. RhoA activity is determined by the amount of GST-RBD-bound RhoA (RhoA-GTP) normalized to total RhoA in whole cell lysates. (C) Quiescent VSMCs were stimulated (FCS) or not (C) with 10% FCS. After 5 minutes, cells were harvested and processed for pull-down experiments. RhoA activity is determined as described above (B).