Fig. 1. Affinity precipitation of cellular GTP-Rho by pull-down assay. (A) CD8 cells were left untreated (control), stimulated with forskolin (10^–4 M, 15 minutes) or incubated with C. difficile toxin B (200 ng/ml, 2 hours), which inhibits all proteins from the Rho family. The lysates from each condition were incubated with 20-30 µg of GST-RBD (Rho binding domain of rhotekin) conjugated with glutathione-Sepharose 4B beads. GTP-Rho was precipitated by centrifugation and subjected to western blotting analysis with anti-RhoA antibodies. Results are representative of three independent experiments with similar results. (B) Densitometric profile (mean values±s.e., n=3) of the GTP-RhoA band relative to control. *P<0.001.