FIG 5.
A monoclonal antibody to RhoA detects modification of this small GTPase by TcdBNAP1 but not by TcdBNAP1V. (A) Recombinant purified RhoA was incubated with TcdBNAP1 and TcdBNAP1V in the presence of UDP-glucose. The preparations were separated by SDS-PAGE and detected by Coomassie blue staining. Parallel samples were transferred to PVDF membranes and developed by Western blotting (WB) using the monoclonal antibody to RhoA. (B) HeLa cells, 3T3 fibroblasts, and Vero cells were treated for the indicated times with TcdBNAP1 or TcdBNAP1V. Cell lysates of treated cells and nontreated control cells were separated by SDS-PAGE, transferred to PVDF membranes, and revealed with the monoclonal antibody to RhoA by Western blotting. As a loading control, membranes were also revealed with a monoclonal antibody to Cdc42.