Figure 7

MEF-2 from myotube nuclear protein extracts binds to the wild-type ART1 promoter A/T-rich element. Gelshift assays were performed by incubation of the [α-³²P]dCTP labelled probe containing the A/T -rich element (WT) of the ART1 gene promoter with nuclear protein extracts (NE) of C3H-10T 1/2 (MyoD-Mb, Mt) and C2C12 (Mb, Mt) cells. The specificity of the protein binding at the A/T-rich element was assessed by the use of a labelled probe containing a mutated ART1 A/T-rich sequence (Mut). For supershift assays, reaction mixtures were preincubated with antibodies (Ab) against MEF-2 (αMEF-2) or a non-specific isotype (ISO) as a negative control. Specific retarded protein/probe complexes are marked by white arrows and supershifted complexes by black arrows. As a positive control a probe containing an MEF-2 binding box of the N-10 gene promoter [25] was used.