Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA

C. M. Silva, D. B. Tully, L. A. Petch, C. M. Jewell, J. A. Cidlowski

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include (i) comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; (ii) localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0°C and in the nucleus and cytosol of cells incubated at 37°C; (iii) binding of the MMTV DNA to highly purified receptor; and (iv) absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.

Original languageEnglish (US)
Pages (from-to)1744-1748
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number7
DOIs
StatePublished - Jun 11 1987

Fingerprint

Glucocorticoid Receptors
Mouse mammary tumor virus
DNA
Proteins
Collodion
Cytosol
Glucocorticoids
Buffers
Gels
Virus Attachment
Mesylates
Cell Extracts
Cell Nucleus
Immobilization
Dexamethasone
Urea
Electrophoresis
Milk
Steroids

ASJC Scopus subject areas

  • General

Cite this

Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA. / Silva, C. M.; Tully, D. B.; Petch, L. A.; Jewell, C. M.; Cidlowski, J. A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, No. 7, 11.06.1987, p. 1744-1748.

Research output: Contribution to journalArticle

@article{89782a2b130f466696d74cc15cc63979,
title = "Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA",
abstract = "To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5{\%} nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include (i) comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; (ii) localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0°C and in the nucleus and cytosol of cells incubated at 37°C; (iii) binding of the MMTV DNA to highly purified receptor; and (iv) absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.",
author = "Silva, {C. M.} and Tully, {D. B.} and Petch, {L. A.} and Jewell, {C. M.} and Cidlowski, {J. A.}",
year = "1987",
month = "6",
day = "11",
doi = "10.1073/pnas.84.7.1744",
language = "English (US)",
volume = "84",
pages = "1744--1748",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "7",

}

TY - JOUR

T1 - Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA

AU - Silva, C. M.

AU - Tully, D. B.

AU - Petch, L. A.

AU - Jewell, C. M.

AU - Cidlowski, J. A.

PY - 1987/6/11

Y1 - 1987/6/11

N2 - To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include (i) comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; (ii) localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0°C and in the nucleus and cytosol of cells incubated at 37°C; (iii) binding of the MMTV DNA to highly purified receptor; and (iv) absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.

AB - To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include (i) comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; (ii) localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0°C and in the nucleus and cytosol of cells incubated at 37°C; (iii) binding of the MMTV DNA to highly purified receptor; and (iv) absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.

UR - http://www.scopus.com/inward/record.url?scp=3042894419&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3042894419&partnerID=8YFLogxK

U2 - 10.1073/pnas.84.7.1744

DO - 10.1073/pnas.84.7.1744

M3 - Article

C2 - 3031647

AN - SCOPUS:3042894419

VL - 84

SP - 1744

EP - 1748

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 7

ER -