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 Machida

Research output: Contribution to journalArticle

1 Citation (Scopus)

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

Fingerprint

Glucocorticoid Receptors
Immunoprecipitation
Adrenergic Receptors
Assays
Response Elements
Genes
Glucocorticoids
Rats
Electrophoretic mobility
Electrophoretic Mobility Shift Assay
Tyrosine Transaminase
3T3 Cells
Baculoviridae
Experiments
Cells
Antibodies
DNA
Proteins

ASJC Scopus subject areas

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

Cite this

UV cross-linking/immunoprecipitation assay : Glucocorticoid receptor-adrenergic receptor gene sequence interaction. / Bai, Ying; Kirigiti, Philbert; Li, Xiaorong; Li, Biao; Tian, Li; Ma, Mark Y J; Machida, Curtis.

In: BioTechniques, Vol. 35, No. 1, 01.07.2003, p. 100-111.

Research output: Contribution to journalArticle

Bai, Ying ; Kirigiti, Philbert ; Li, Xiaorong ; Li, Biao ; Tian, Li ; Ma, Mark Y J ; Machida, Curtis. / UV cross-linking/immunoprecipitation assay : Glucocorticoid receptor-adrenergic receptor gene sequence interaction. In: BioTechniques. 2003 ; Vol. 35, No. 1. pp. 100-111.
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