Electrophoretic mobility shift assays (EMSA) and transient transfection of site-directed mutagenesis constructs. (A) EMSA results using radiolabelled probes for the mouse and human AT2 and CArG box regions with C2C12 nuclear extract. The mouse AT2 region showed strong binding of a protein that was supershifted by a myocyte enhancer factor 2 (MEF2)-specific antibody. The human probe did not bind this protein, but did bind a protein that was supershifted by an octamer binding transcription factor 1 (OCT1)-specific antibody. The mouse CArG probe demonstrated strong binding to a protein that was supershifted by a serum response factor (SRF)-specific antibody while the human probe did not bind this protein. (B) Reporter plasmid transfection results indicated that targeted mutagenesis of the human CArG box motif (Human 1.0 kb mouse CArG) increased activity of the human MyHC-IIb promoter construct ~fivefold in mouse cells and ~twofold in human cells, resulting in activity equal to that of the full-length mouse construct. Conversely, replacing the mouse CArG box sequence with the corresponding human sequence significantly reduced activity of the mutated mouse construct (Mouse 1.0 kb human CArG) in both mouse and human cells. Mutagenesis of the human AT2 region (Human 1.0 kb mouse AT2) increased activity ~twofold in mouse cells but had no effect in human cells. Values (mean ± standard error or mean) represent the result of at least three duplicate experiments and are expressed as fold-activity relative to the mouse 1.0 kb MyHC-IIb reporter plasmid. *P < 0.05 Human 1.0 kb mouse AT2 or mouse CArG versus Human 1.0 kb; **P < 0.05 Mouse 1.0 kb human CArG versus Mouse 1.0 kb.