Fig. 5. Tbx1 activates the Pitx2 enhancer (Pitx2-ASE) by binding to a T-half site within the enhancer and through interaction with Nkx2.5. (A) Luciferase assays show that Tbx1 or Nkx2.5 can activate the Pitx2-ASE reporter weakly. Co-transfection of Tbx1 and Nkx2.5 leads to a 12-fold activation of the Pitx2-ASE reporter. Data are presented as mean±s.e.m. for three independent experiments. (B) Luciferase assays using the Pitx2 enhancer mutated at the T-half site (Pitx2-Mut) do not show activation of Pitx2-Mut when transfected with either Tbx1, Nkx2.5 or both expression constructs. Data are presented as mean±s.e.m. for three independent experiments. (C) In EMSAs, Tbx1 binds to the putative T-half site found within the Pitx2 enhancer (wild type). Arrow shows wild-type Tbx1-DNA complex. Addition of Tbx1 antibody results in a supershift of the complex, marked by an arrowhead. Mutation of the T-half site (M1, M2) does not lead to the formation of a Tbx1-DNA complex. Binding of Tbx1 to the consensus T-half site (Cons) is weak. (D) Co-immunoprecipitation of co-transfected Flag-Nkx2.5 and Tbx1-GFP followed by detection of Tbx1 (arrow, upper blot) and Flag protein (arrow, bottom blot) shows interaction between Nkx2.5 and Tbx1. Untransfected cells, and cells transfected with only Tbx1 or Nkx2.5 were used as controls. Unspecific bands (upper blot) and IgG bands (bottom blot) are marked with an arrowhead.