FIG. 1.

Myocardin and GATA4 synergistically activate the Nkx2.5 enhancer. (A) Primary neonatal cardiomyocytes were infected with adenoviruses encoding myocardin or lacZ (as a negative control). Four days later, expression of Nkx2.5 transcripts was measured by semiquantitative reverse transcription-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were detected as a loading control. (B) Gel mobility shift assays were performed with a radiolabeled oligonucleotide corresponding to the CArG box in the Nkx2.5 enhancer and in vitro-translated SRF and myocardin proteins, as indicated. Myocardin formed a stable ternary complex with SRF on this sequence (lane 3). Anti-SRF antibody supershifted the SRF complex (lane 2), and anti-Flag antibody supershifted the ternary complex formed by SRF and Flag-myocardin (lane 4). (C) COS cells were transiently transfected with the luciferase reporter plasmids shown above each panel and the indicated amounts (in nanograms) of myocardin expression vector. Luciferase activity was assayed and is expressed as fold activation above the level of expression of the reporter gene alone. (D) COS cells were transiently transfected with the luciferase reporter plasmids shown above each panel and the indicated amounts (in nanograms) of myocardin expression vector with and without a GATA4 expression vector (100 ng), and luciferase activity was determined as in panel C.