Identification of Rho GEF and RhoA Activation by Pull-Down Assays

The small GTPase RhoA participates in actin and microtubule machinery, cell migration and invasion, gene expression, vesicular trafficking and cell cycle, and its dysregulation is a determining factor in many pathological conditions. Similar to other Rho GTPases, RhoA is a key component of the wound-healing process, regulating the activity of different participating cell types. RhoA gets activated upon binding to guanine nucleotide exchange factors (GEFs), which catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). GTPase-activating proteins (GAPs) mediate the exchange of GTP to GDP, inactivating RhoA, whereas guanine nucleotide dissociation inhibitors (GDIs) preserve the inactive pool of RhoA proteins in the cytosol. RhoA and Rho GEF activation is detected by protein pull-down assays, which use chimeric proteins with Rhotekin and G17A mutant RhoA as “bait” to pull down active RhoA and RhoA GEFs, respectively. In this chapter, we describe an optimized protocol for performing RhoA and GEF pull-down assays.