FIG. 5.

Rhotekin pulldown assay. Lysates and cytosols of CNF1-treated (400 ng of CNF1/ml, 3 h) HeLa cells were incubated with and without YopT (5 μg/sample) for 30 min at 37°C. After incubation with YopT, 1/15 of the volume was taken as input control shown in the lower panel. The rest of the lysates and cytosols were incubated with GST-rhotekin beads for 60 min at 4°C. After washing, rhotekin-bound RhoA and the input control were separated by SDS-PAGE and detected by Western blotting with a RhoA-specific antibody. After blotting, it was checked by Ponceau staining that equal amounts of beads have been used for precipitation. Shown is a typical result of three independent experiments.