Sp1 binding to the 34-nt segment in vitro. (A) EMSA was performed in the presence of increasing amounts of recombinant Sp1 mixed with 32P-labeled oligonucleotide. (lane 1: 20 ng, lane 2: 30 ng, lane 3: 50 ng, lane 4: 100 ng, lane 5: 200 ng) (B) Nuclear extracts were prepared from HepG2 cells and incubated with biotinylated oligonucleotide derived from the 34-nt region of the edn promoter. The DAPA samples were separated by 8% SDS-PAGE and immunoblotted for Sp1. Relative densitometric quantitation of bands in lanes 1 to 5 are as follows: biotin control, wild type, mutant -62/-48, mutant -74/-65, mutant -62/-48 & -74/-65. The difference between the two groups is statistically significant (P < 0.05), as determined by the Wilcoxon Rank Sum test.