Rho GTPase activation assay

RhoA activity was assessed with the Rhotekin binding assay as described previously (Cytoskeleton, Inc., Denver, CO). Briefly, cells were grown in 100-mm dishes. After serum starvation for 24 hours, cells were treated either with calpeptin (Rho Activator I; 0.1 mg/ml final) for 30 minutes or with carrier only (DMSO). Cell lysates were incubated with the GST-fused Rho-binding domain of Rhotekin immobilized on reduced glutathione-agarose for 2 hours at 4°C in a tube rotator and then rinsed. The level of active RhoA and total RhoA was detected by Western blotting with anti-RhoA antibody.