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Research Article

Site-Specific Anti-C-ERB A Antibodies Recognizing Native Thyroid Hormone Receptors: Their use to Detect the Expression and Localization of α and β C-ERB A Proteins in Rat Liver

, , , , , , , , & show all
Pages 201-215 | Published online: 26 Sep 2008
 

Abstract

The cell-specific expression and tissue distribution of c-erbA proteins α and β is still unknown. To address this problem, we prepared anti-peptide antibodies directed against epitopes of human (h) c-erbA, specific for the α or β form of thyroid hormone receptors. The cDNAs coding for h c-erbA β1, α1 and α2 were transcribed and the mRNAs were translated in vitro in the presence of 35S-methionine, and then their reactivity with the antisera was evaluated. The antiserum anti-β 62–81 immunoprecipitated only the β1 receptor. The antiserum anti-α 144–162 determined precipitation of both α1 and α2 proteins but not of the β1 receptor. Anti-α2 431–451 produced a selective precipitation of α2, and had no effect on α1 or β1 receptor. In order to study the interaction of the antibodies with native T3 receptor we evaluated the binding of antibodies to rat liver T3 receptors by Sephacryl S300 chromatography: both antisera anti-β 62–81 and anti-α 144–162 caused a partial shift of the labeled T3–receptor complex to a higher molecular form, while the antibody directed against c-erbA α2 did not produce any significant shift. The anti-peptide antibodies were then immunopurified by affinity chromatography and used to immunolocalize the different forms of c-erb A proteins in adult and fetal rat liver, by a sensitive immunohistochemical technique. All 3 antibodies stained mainly the nuclei of the majority of adult liver cells. No staining was detectable when the original antiserum was deprived of anti-peptide antibodies by running through the affinity columns or when the antibodies were pre-absorbed with the homologous peptide. No significant staining was present in the liver from rat fetus.

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