Fig. 5. Tsh proteins are CtBP-dependent repressors. (A) Deletion of the CtBP-interaction motif in Tsh abolishes the interaction with CtBP in vitro (bottom) and fails to suppress mod expression in the labial segment of 69B>tshPLDLS embryos (top, compare with Fig. 3E). (B) pABgal-mouse Tsh1, 2, and 3 (0.025 and 0.1 µg) alone repress pGL2-5xUAS-Luc reporter activity (0.25 µg). (C) GST-pull down assay showing the interaction between in vitro translated mouse Ctbp1 and GST-mouse Tsh fusion proteins. Non-specific interaction is shown with GST alone. (D) In an independent experiment from B, co-expression of 0.1 µg mouse Ctbp1 with 0.1 µg Gal4-mouse Tsh fusions leads to a synergistic inhibition. Deletion of the putative CtBP interaction motif in pABgal-mouse Tsh1 reduces the activity of the reporter from 5 to 2.5-fold and abolishes the synergistic repression in the presence of mouse Ctbp1. Mouse Ctbp1 alone does not affect the reporter activity (not shown). Data are presented as fold repression relative to luciferase activity, normalized to the ß-galactosidase activity, of the reporter in presence of empty pABgal and pcDNA3.myc.