Fig. 1. EphrinA1 represses spreading of VSMCs on laminin. (A) Activation of EphA2 by immobilized ephrinA1. Quiescent VSMCs plated on tissue culture dishes coated with laminin (0.25 µg/cm²) and 1 µg/cm² of either Fc or ephrinA1 for the indicated times were lysed as described. EphA2 was precipitated (IP) with an anti-EphA2 antibody. Immunoblotting was performed using an anti-phosphotyrosine to determine the activation of EphA2 (Phospho-EphA2) or an anti-EphA2 to control loading. The fold induction is calculated from densitometric measurements of phosphotyrosine signal normalized to EphA2 loading in lysates of ephrinA1-stimulated cells compared with Fc control at each time point. (B) Representative phase-contrast microscopy of quiescent VSMCs allowed to spread for 16 hours on tissue culture dishes coated with laminin (0.25 µg/cm²) and the indicated amounts of control Fc fragment (L+Fc) or ephrinA1/Fc (L+A1). Reversion of the effect was initiated by trypsinizing VSMCs attached on dishes coated with laminin and 1 µg/cm² of either Fc or A1, seeding them on another dish coated with laminin alone and allowing spreading for 24 hours. Bar, 40 µm. (C) Quiescent VSMCs were allowed to spread for the indicated times 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). Bar, 40 µm.

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