Figure 6

Chromatin immunoprecipitation assay performed on differentiating C2C12 cells to evaluate in vivo MyoD binding at the ms1 promoter. (A) A schematic map of the amplified DNA fragments (product size) and the primer locations encompassing E1, E2 and E3. TSS position is also illustrated. Proteins were cross-linked to the DNA (in C2C12 cells during myogenic differentiation) with formaldehyde, DNA was sheared by sonication, and Abs directed against IgG or MyoD were added to precipitate any protein-DNA complexes. The precipitated complexes were pre-cleared with protein A beads. Samples analyzed included proliferating, subconfluent myoblasts (M), confluent myoblasts harvested prior to the induction of differentiation (D0), myoblasts subjected to differentiation conditions for 24 hours (D1), and differentiating myotubes at 48 hours (D2) or 72 hours (D3) post-differentiation. Quantitative real time PCR were performed on isolated DNA using primers encompassing the proximal (B) and distal (C) E-Box sequences (E2/E3 and E1 respectively). Amplification was quantified and normalised to the input of each sample. The results are expressed as mean ± SE of at least three different ChIPs. Statistically significant differences in fold enrichment are indicated by *P < 0.05. Representative PCR reactions were stopped in the linear amplification range and run on agarose gel for visualisation.