Fig. 8. Ephrin-A5 stimulation of EphA3-expressing 293T cells mediates a rapid increase of active Rho A. The levels of active RhoA in parental (B) or EphA3-293T cells (A,C-E) transfected with w/t Crk (D) or SH3^* Crk (E) as indicated, and which had been treated as adherent (A,B,D) or suspension (C) cultures with preclustered ephrin-A5, were analysed at the indicated times by precipitating GTP-bound RhoA with GST-RBD. Precipitated proteins and aliquots of the total lysates were analysed by immunoblotting with an anti-RhoA Mab. The intensity of bands representing active RhoA (RhoA-GTP) was normalised by comparison with Rho levels in corresponding total lysates using densitometry, and is indicated relative to baseline levels (given the arbitrary value `1') in the absence of ephrin-A5 stimulation. Each result represents the mean and s.d. from a minimum of three independent experiments.