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. 2009 Jun 4;284(31):20593–20601. doi: 10.1074/jbc.M109.021477

FIGURE 3.

FIGURE 3.

DLX5 binds to MYC promoter in vitro and in vivo. A, gel shift assays were performed using recombinant human DLX5 protein and radiolabeled oligonucleotides. DLX5 binding could be competed by excess molar concentrations (25-, 50-, or 250-fold excess) of unlabeled probes, as indicated by the triangles. Supershift was observed by adding a DLX5-specific antibody (α-DLX5) into each reaction. The arrow indicates oligonucleotides binding to DLX5. B, ChIP assay carried out using ChIP-grade antibody against DLX5 (or normal goat IgG as a negative control) with HeLa cells transiently transfected with DLX5 or NCI-H322M cells. PCR products represent a portion of the MYC promoter (MYC). The upstream control (Upstream Con) represents a portion of the upstream MYC promoter containing nucleotides −2305 to −2015. The second control (Downstream Con) represents a downstream sequence containing nucleotides 1801–2062. Nucleotide numbers are relative to the P2 transcription start site (+1) in the MYC promoter. CON, positive control using total genomic DNA as template; DLX5, PCR products amplified from the ChIP samples mediated by anti-DLX5 antibody; IgG, absence of PCR product corresponding to MYC promoter in sample mediated by IgG.