MSC dimers have relaxed requirements for flanking sequence compared to MyoD dimers. (A) MyoD homodimers and MyoD:E-protein heterodimers do not bind well to MSC-specific sequences, but bind after a small number of sequence changes. Electrophoretic mobility shift assays (EMSAs) were performed using in vitro translated proteins and probes as indicated. The asterisks indicate the location of what are, judging by their relative mobility, small amounts of E-protein homodimers. (B) MSC heterodimers can be competed off a preferred binding site equally well by competitors with variations in their flanking sequence, while MyoD heterodimers cannot. MyoD:E and MSC:E heterodimers were subjected to competition by excesses of cold probes as indicated. 25× and 50× refer to the excess mass of cold probe relative to hot probe. Variations in competitor sequences are indicated, and ‘CG Ebox’ refers to a probe with an inverted central dinucleotide sequence that abolishes all binding of MyoD and MSC. (C) Single nucleotide changes in flanking sequence can completely abrogate MyoD dimer binding, but still be permissive of MSC dimer binding. Shift assays were performed using proteins and probes as indicated. Each type of dimer combination was run in two lanes, with one lane having a probe with ‘A’ in the −2 position relative to the E-box, and the other lane having a probe with ‘T’ in that position, as indicated in red. All shifts were performed using a sufficient excess of probe so that visible free probe was present for all lanes (not shown in 4A and 4B). All probe counts were quantitated before addition to ensure there were roughly equivalent amounts in all compared lanes. Negative control lanes indicate lanes where probes were tested with an in vitro translated empty CS2 vector to identify any non-specific binding. EMSA, electrophoretic mobility shift assay; MSC, musculin.