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

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — A, sequence homology between the consensus C/EBP binding site and −1600 region of the hAGT gene promoter. Line 1, consensus C/EBP binding site; line 2, nucleotide sequence located between −1596 and −1613 of the hAGT gene; line 3, oligonucleotide sequence containing mutated C/EBP binding site. B, ChIP assay shows that C/EBP binds to the −1600 region of the hAGT gene promoter in HepG2 cells. The ChIP assay was performed as described in the legend to Fig. 6B except that C/EBPβ antibody was used for immunoprecipitation. Shown are PCR product obtained in the presence of C/EBPβ antibody (lane 1), PCR product in the presence of preimmune serum (lane 2), PCR product obtained in the presence of C/EBPβ antibody from another region of the promoter that does not contain C/EBP site (lane 3), and PCR product obtained from the genomic DNA without immunoprecipitation (positive control) (lane 4). C, EMSA confirms that nucleotide sequence located between −1596 and −1613 of the hAGT gene promoter competes with the C/EBP binding site. EMSA was performed using radiolabeled oligonucleotide containing the consensus C/EBP binding site in the presence of HepG2 extract and competed with an oligonucleotide containing either wild type or mutated C/EBP site located in the −1596 to −1613 region of the hAGT gene promoter. Lane 1, complex obtained in the presence of radiolabeled oligonucleotide containing consensus C/EBP site; lane 2, competition with a 20-fold excess of cold C/EBP consensus oligonucleotide; lanes 3–6, competition in the presence of a 50- and 100-fold excess of an oligonucleotide containing either wild type or mutant C/EBP sequence from the hAGT gene; lane 7, reaction in the presence of C/EBPβ antibody; lane 8, reaction in the presence of preimmune serum (PIS). D, EMSA confirms that nucleotide sequence located between −1596 and −1613 of the hAGT gene promoter binds to C/EBPβ. EMSA was performed using radiolabeled oligonucleotide containing the C/EBP site located between −1596 and −1613 of the hAGT gene promoter in the presence of HepG2 extract. Lane 1, complex obtained in the presence of radiolabeled oligonucleotide alone; lanes 2–5, competition in the presence of a 50- and 100-fold excess of wild type and mutant cold C/EBP oligonucleotides; lane 6, reaction in the presence of preimmune serum; lane 7, reaction in the presence of C/EBPβ antibody; lane 8, reaction in the presence of a 50-fold excess of cold consensus C/EBP oligonucleotide. E, mutation of C/EBP site located between −1596 and −1613 of the hAGT gene promoter reduces glucocorticoid-induced promoter activity. Reporter constructs containing 1618 bp of the hAGT gene promoter with either wild type or mutated C/EBP site (located between −1596 and −1613) were transiently transfected in HepG2 cells, and glucocorticoid-induced promoter activity was analyzed as described above. The empty box shows glucocorticoid-induced promoter activity of the wild type construct, and the filled box shows glucocorticoid-induced promoter activity of the mutated construct. Error bars, S.E.