Identification of novel RHOA interacting patners

RHO subfamily of small GTPases comprise highly conserved family members RHOA, RHOB, and RHOC which cycle between GTP-bound 'active' and GDP-bound 'inactive' states. In the active form, RHO proteins interact with a variety of downstream effector proteins, controlling their activity and function. Many of the RHO subfamily effector proteins such as ROCK, PKN, and mDIA, are key regulators of actin cytoskeleton and cell motility. To identify novel effector proteins for RHOA,we carried out a GST-pulldown from heavy or light SILAC labelled HeLa cells using GST tagged GTP-bound RHOA, or GST alone control, as bait. Pulldowns were performed in duplicates with switched labellings. Specific interactors were destinguished from the background on the basis of SILAC Heavy to Light ratios between GST-RHOA and GST alone pulldowns.