Fig. 5. A MEF2-binding site is required for cardiac expression of Bop (A) The sequence of the mouse Bop gene containing the essential MEF2 site and the E-boxes is shown along with homology to the corresponding regions from other species. (B) 5' flanking sequences of mouse Bop genes showing conserved MEF2 site at –329/-320. (C) A gel mobility shift assay was performed using nuclear extracts from COS-1 cells transfected with a MYC-MEF2C expression plasmid and the radiolabeled MEF2 site shown in panel A as probe. Specific and nonspecific competitors were used at 50-fold molar excess. Antibody supershift used 1 µg of polyclonal anti-MYC antibody. (D,E) MEF2 site is essential for Bop expression in the anterior heart field in vivo. F0 transgenic mouse embryos were generated using constructs 1, 2, 5 and 6 (see Fig. 2B), and stained for expression of ß-galactosidase at E11.5. Mutation of the MEF2 site abolished expression in the anterior heart field without affecting expression in skeletal muscle. Four independent F0 transgenic embryos were analyzed with construct 5 (D) and five independent embryos were analyzed for construct 6 (E). All embryos with each transgene showed comparable expression patterns.