Fig. 3. DNA binding and transactivation analysis of the Nkx2-5Y-A mutant protein. (A) EMSA comparing DNA binding of in vitro-translated Nkx2-5 and Nkx2-5Y-A using an Nkx2-5-binding oligonucleotide probe. Anti-Nkx2-5 antibody and specific and non-specific competitor oligonucleotides were used to assess binding specificity. (B,C) In vitro transcriptional analysis using FLAG-tagged Nkx2-5 and Nkx2-5Y-A expression vectors and a multimerised Nkx2-5-binding site synthetic enhancer-luciferase reporter (B) or with and without Gata5 and Tbx20a on the Nppa (–700) and Gja5 (–1190 to +121) promoter-luciferase reporters in C3H 10T1/2 cells (C). Inset in B shows western blot of transfected proteins. Significance assessed using Student's t-test.