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. 2010 Oct 26;285(52):41172–41186. doi: 10.1074/jbc.M110.167585

FIGURE 2.

FIGURE 2.

A, nucleotide sequence around −1670 has homology with the HNF-1 binding site. Line 1 shows the consensus HNF-1 binding site, line 2 shows the nucleotide sequence containing −1670A, and line 3 shows the nucleotide sequence containing −1670G. B, ChIP assay shows that nucleotide sequence around −1670 binds to HNF-1α in HepG2 cells. Immunoprecipitated DNA from HepG2 cells was used to amplify a 203-bp sequence from the hAGT gene promoter (−1546 to −1748) containing the HNF-1α binding site. Lane 1, PCR product obtained in the presence of HNF-1α antibody; lane 2, PCR product obtained in the presence of preimmune serum; lane 3, PCR product obtained in the presence of HNF-1α antibody from another region of the promoter that does not contain the HNF-1 site; lane 4, PCR product obtained from the genomic DNA without immunoprecipitation (positive control) (lane 4). C, EMSA shows that nucleotide sequence containing −1670A has stronger homology with the HNF-1α binding site as compared with −1670G. EMSA was performed using radiolabeled oligonucleotide containing −1670A in the presence of HepG2 nuclear extract. Lane 1, radiolabeled oligonucleotide containing −1670A without competitor; lane 2, competition with a 50-fold excess of cold oligonucleotide containing consensus HNF-1 binding site; lane 3, competition with a 50-fold excess of cold oligonucleotide containing −1670A; lane 4, competition with a 50-fold excess of cold oligonucleotide containing −1670G; lane 5, competition with a 50-fold excess of cold oligonucleotide containing nonspecific oligonucleotide; lane 6, reaction in the presence of HNF-1α antibody; lane 7, reaction in the presence of preimmune serum (PIS).