FIGURE 5.

MyoD binds to E-boxes of the SelW promoter in differentiating C2C12 cells. A, schematic representation of SelW promoter −973SelW/Luc harboring four putative E-boxes. B, SelW promoter activity of −973SelW/Luc and E-box mutant plasmids in differentiating C2C12 cells. C2C12 cells were transfected with SelW promoter −973SelW/Luc or E-box mutants and pRL-TK. Luciferase activity was measured by harvesting of transfected C2C12 cells after 1 day of culture in DM. Data are expressed as means ± S.D. of three or four independent experiments in triplicate. C and D, EMSA of the putative E-boxes in the SelW promoter. [γ- ³²P]dATP-labeled oligonucleotide probes for the consensus E-boxes were incubated with nuclear extracts (NE) of differentiating C2C12 cells in DM for 1 day. For supershift analysis (C), anti-MyoD antibody was incubated with nuclear extracts prior to addition of labeled probes. For competition assays of the E1 and E3 E-boxes (D), unlabeled competitors were added (10-, 50-, and 100-fold excess) in a dose-dependent manner to nuclear extracts prior to incubation with labeled probes. The unlabeled competitor MCK was the same oligonucleotide used in C, and E1m was the oligonucleotide used for the mutagenesis of E1.