CtBP reverse two-hybrid system. (a) CtBP expression as fusions with the 434 bacteriophage DNA binding protein is induced by IPTG. The CtBP-434 fusion proteins associate to form a functional repressor that prevents expression of the reporter genes HIS3 and KanR, inhibiting growth on selective media. The third reporter gene, LacZ is used to quantify the protein–protein interaction by o-nitrophenyl-β-d-galactoside (ONPG) assays. (b) ONPG assay of the CtBP1, CtBP2 and CtBP2G189A RTHS. (c) Drop-spotting serial dilutions (2.5 μL of ~10n cells per mL) of the CtBP1, CtBP2, CtBP2G189A, and ATIC (positive control)41 RTHS onto selective media plates with and without IPTG. Data shows formation of a functional repressor in all cases except the dimerization-incompetent CtBP2G189A RTHS.