Activation assay for RhoA and Cdc42.

The activation of RhoA or Cdc42 was determined with pulldown assays for activated RhoA or activated Cdc42, as previously described (87), using RhoA and Cdc42 activation assay kits (Cell Biolabs) as directed by the manufacturer's instructions. For the analysis of RhoA activation, cell lysates were incubated with Rhotekin RBD agarose beads, which have a high affinity for GTP-RhoA. For the analysis of Cdc42 activation, cell lysates were incubated with PAK1 PBD agarose beads, which have a high affinity for GTP-Cdc42. Affinity-precipitated activated GTP-bound RhoA or Cdc42 levels were then analyzed by immunoblotting using RhoA and Cdc42-specific antibodies.