Figure 3

Transcription factor Sp1 and Egr-1 bind specifically to the Sp1/Egr-1 overlapping site in NGX6 minimal promoter. (A) Electrophoretic mobility shift assay demonstrating complex formation between the G/C-rich, potential Sp1/Egr-1 binding domain of NGX6 gene promoter in HT-29 cells. Oligonucleotides -54/-39 containing a Sp1/Egr-1 overlapping site was incubated with 10-μg nuclear extracts of HT-29 cells in EMSA. For competition assay, 5-pmol of unlabeled wild-type or mutated oligonucleotide was added to the reaction mixture, before the addition of labeled probes. (B) ChIP assay of the NGX6 promoter at the Sp1/Egr-1 overlapping binding site. Crosslinked chromation was sheared with nine 10-sec pulses at 10-sec intervals. Immunoprecipitation of chromatin fragments were carried out with anti-Sp1, and anti-Egr-1, respectively. Rabbit IgG was used as a negative control and Input was used as a positive control. After de-crosslinking, the purified DNA samples were analyzed by PCR. water (a reaction without any DNA) were used as a negative control for PCR.