To generate N-terminal hemagglutinin (HA) epitope-tagged RHOA variants, the cDNA sequence encoding human full-length RHOA was cloned into the pCDH-HA mammalian lentivirus vector as we described previously (23 (link)). To generate RHOA fusion constructs for E.coli recombinant protein purification, the human RHOA cDNA sequence was cloned into either the pPRO-TEV-His or pGEX-4T1 bacterial expression vectors that add amino-terminal His6 or glutathione S-transferase (GST) tags, respectively (23 (link)). Site-directed mutagenesis using the Q5 Site-directed Mutagenesis Kit (NEB) was done according to manufacturer’s guidelines to generate cDNA sequences encoding RHOA mutants T19N, Y42C, L57V and Q63L. The cDNA sequences encoding the human ECT2 GEF catalytic PH-DH domain (residues 406–777) were subcloned into the pPRO-TEV-His vector and the RHO binding domain (RBD) of human ROCK1 (residues 947–101) in a pGEX4T1 vector were generated as described previously (23 (link)). The plasmid pEGFP-RhoGDI1 encoding full-length human RhoGDI1 was obtained from Mark Philips (New York University, USA) and pGEX2T1-Rhotekin-RBD (mouse, residues 7–89) from Keith Burridge (University of North Carolina at Chapel Hill, USA). The plasmids pGEX4T1-mDia1-RBD (mouse, residues 69–451) and pGEX4T1-p190RhoGAP (human, residues 1250–1531, GAP domain) were provided by Reza Ahmadian (Heinrich-Heine University, Düsseldorf, Germany). COS-7 cells (RRID:CVCL_0224) and HEK293T cells (RRID:CVCL_0063) were obtained from the American Type Culture Collection (ATCC) and NIH/3T3 mouse fibroblasts (RRID:CVCL_0594) were provided by Geoffrey Cooper (Dana-Farber Cancer Institute, Boston, MA). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (HEK293T, COS-7) or Colorado calf serum (NIH/3T3), penicillin, and streptomycin. Cells were passaged for one month or 10 passages a humidified chamber with 5% CO2 at 37°C. Cell lines were monitored regularly for mycoplasma contamination using the Lonza MycoAlert Mycoplasma Detection Kit.