Figure 8

Binding of Sp1 and MAZ to the 34-nt region in the edn promoter in vivo. (A) Cells were fixed by 1% formaldehyde, and the genomic DNA was sonicated until the size was approximately 1000 bp. The samples were then analyzed by the ChIP assay for Sp1, using IgG as a negative control. The samples were subjected to 30 cycles of PCR and separated on a 2% agarose gel. (B) ChAP assays were performed using HepG2 cells transfected with pcDNA3/MAZ-6H, pcDNA3/MAZΔF34-6H, or empty pcDNA plasmid. Two days after transfection, cells were scraped, cross-linked and sonicated, and the DNA-protein complexes were incubated with nickel resin slurry for 1 hr at 4°C. The affinity precipitated DNA/protein complexes were eluted by 1 M imidazole and subjected PCR amplification. Input DNA served as a positive control.