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PMID: 1655807 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Retinoic acid receptor gamma: specific immunodetection and phosphorylation.

The Journal of cell biology ·Vol. 115 ·No. 2 ·1991-10-00 ·Pages 535-45

Rochette-Egly C, Lutz Y, Saunders M, Scheuer I, Gaub MP, Chambon P

Abstract

Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor gamma 1 (hRAR-gamma 1 and mRAR-gamma 1, respectively) were used to generate anti-RAR-gamma 1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1 gamma (A1)), region F (Ab2 gamma (mF) and Ab4 gamma (hF)) and region D2 (Ab5 gamma (D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-gamma 1 proteins in COS-1 cells transfected with expression vectors containing the RAR-gamma 1 cDNAs. They all reacted with both human and mouse RAR-gamma 1 proteins, except Ab4 gamma (hF) that was specific for hRAR-gamma 1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-gamma 1 F region were also obtained (RP gamma (mF)) and found to be specific for mouse RAR-gamma 1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-gamma 2 isoform which differs from RAR-gamma 1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-gamma 1 (Ab1 gamma (A1)) only reacted with the RAR-gamma 1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-gamma 1 and gamma 2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-gamma 1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.

MeSH Terms
Alkaline Phosphatase/metabolism Amino Acid Sequence Animals Antibodies, Monoclonal Blotting, Western Carrier Proteins/analysis,immunology,metabolism Cloning, Molecular Embryo, Mammalian/metabolism Humans Mice Molecular Sequence Data Peptide Fragments/immunology Phosphorylation Protein Processing, Post-Translational/physiology Rabbits Receptors, Retinoic Acid Transfection/genetics Tretinoin/metabolism Tumor Cells, Cultured
Chemicals
Antibodies, Monoclonal Carrier Proteins Peptide Fragments Receptors, Retinoic Acid Tretinoin Alkaline Phosphatase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Rochette-Egly C
Laboratoire de Génétique Moléculaire des Eucaryotes du Centre National pour la Recherche Scientifique, l'Institut National de la Santé et de la Recherche Medicale, Faculté de Médecine, Strasbourg, France.
Lutz Y
Saunders M
Scheuer I
Gaub M P
Chambon P
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.115.2.535
Published
1991-10-00
Pages
535-45
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2289155
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.115.2.535 PMC Full Text View journal details
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