UV cross-linking/immunoprecipitation assay: Glucocorticoid receptor-adrenergic receptor gene sequence interaction

Ying Bai, Philbert Kirigiti, Xiaorong Li, Biao Li, Li Tian, Mark Y.J. Ma, Curtis A. Machida

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

The rat β1-adrenergic receptor (β1-AR) gene contains glucocorticoid response element (GRE) half-sites at positions -2767 and -945. In electrophoretic mobility shift assay (EMSA) experiments, neither β1-AR GRE half-site recognized glucocorticoid receptors (GRs) obtained from baculovirus high-level expression systems or from mammalian cells. We have developed a sensitive UV cross-linking/immunoprecipitation assay, using a 524-bp fragment containing the prototypical GRE obtained from the rat tyrosine aminotransferase promoter sequence and using antibodies recognizing mammalian GR. Using this assay, we provide evidence that rat β1-AR gene sequences recognize mammalian GRs expressed in mouse 3T3 cells and that the site of GR interaction does not appear to specifically contain the β1-AR GRE half-sites. This represents one of the first reports demonstrating the utility of a UV cross-linking/immzmoprecipitation assay in the detection of mammalian GR interaction with β1-AR sequences, is consistent with the lack of specific DNA-GR protein complexes observed in EMSA experiments using oligonuticleotide probes containing the β1-AR GRE half-sites, and provides evidence that mammalian GR interaction occurs at complex rat β1-AR gene sequences.

Original languageEnglish (US)
Pages (from-to)100-111
Number of pages12
JournalBioTechniques
Volume35
Issue number1
StatePublished - Jul 1 2003

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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