To verify protein–protein interaction among three Rho GTPases, a yeast two-hybrid assay was performed using the
Saccharomyces cerevisiae L40 strain harboring reporter genes
HIS3 and β-galactosidase under the control of upstream LexA-binding sites. PCR was performed to make a bait vector for RhoA (sense, 5′-GAATTCCTGCCATCC-GTAAGAAA-3′; antisense, 5′-CTGCAGACTGCAGGGCAGCCCGGGT-3′), Rac1 (sense, 5′-GAATTCCAGGCCATCAAGTGCGTG-3′; antisense 5′-CTGCAGTTAGGGGCACAGGACGGCCCG-3′), and Cdc42 (sense, 5′-GAATTCCAGACAATCAAGTGTGTT-3′; antisense, 5′-CTGCAGGGGAGGCTCCAGGGCAGC-3′), using rabbit Rho GTPases cDNA fragments as the templates. The PCR products were subcloned in-frame into the
EcoRI–
PstI site of pBHA (a bait vector containing LexA DNA-binding domain), resulting in the construction of pBHA-RhoA, pBHA-Rac1, and pBHA-Cdc42. Similar subcloning was conducted to construct prey vector for RhoA (sense, 5′-CTCGAGCTGCCATCCGTAAGAAA-3′; antisense, 5′-GAATTCACTGCAGGGCAGCCCGGGT-3′), Rac1 (sense, 5′-CTCGAGAGGCCATCAAGTGCGTG-3′; antisense 5′-GAATTCTTAGGGGCACAGGACGGC-3′), and Cdc42 (sense, 5′-CTCGAGAGACAATCAAGTGTGTT-3′; antisense, 5′-GAATTCGGGAGGCTCCAGGGCAGC-3′), using PCR. The resultant PCR fragments were inserted in-frame into the
XhoI–
EcoRI site of pGAD10 (a prey vector with the Gal4 activation domain) and named pGAD10-RhoA, pGAD10-Rac1, and pGAD10-Cdc42, respectively. The yeast L40 strain was cotransformed with these bait and prey plasmids. Transformation of the yeast strain was performed using the lithium acetate method.
45 Cotransformants were plated on solid YNB (0.67% yeast nitrogen base without amino acids, 2% dextrose) + adenine + histidine medium (YNB+AH) and grown at 30°C. The colonies were replica printed to X-Gal plates at 30°C and checked daily for blue color for 3 days. Colonies that turned blue were selected on YNB lacking histidine (YNB+A)–selective media and then retested in X-Gal plates, to confirm that they produced β-galactosidase, which is selective for interaction among Rho, Rac, and Cdc42.