Fig. 6. Tbx5 and Tbx20 are localized to the nucleus and can activate transcription on the Nppa/ANF promoter. (A) Schematic depicting the amino acid positions of the T-box domains of Tbx5 and Tbx20. (B) Schematic of Rat Nppa/ANF-luciferase reporter construct showing T-box-binding site consensus sequences and their relative position within the promoter relative to translation start site. (C-H) Transfected cells were stained with anti-V5 (C,F; Cy2, green) for TBX5-V5 and TBX20-V5, and with anti-phosphotyrosine (D,G; Cy3, red) to visualize cytoplasmic compartment. Overlaid image of Cy2 and Cy3 staining (E,H). (I) Rat ANF-luciferase co-transfected with a constant amount of Tbx5 (100 ng) and increasing amounts of Tbx20 (25, 50, 100 and 500 ng) in 293T cells, and the level of transcriptional activation is expressed as relative luciferase units based on average of three independent experiments performed in triplicate.