Site-specific anti-c-erb A antibodies recognizing native thyroid hormone receptors: their use to detect the expression and localization of alpha and beta c-erb A proteins in rat liver.
The cell-specific expression and tissue distribution of c-erbA proteins alpha and beta is still unknown. To address this problem, we prepared anti-peptide antibodies directed against epitopes of human (h) c-erbA, specific for the alpha or beta form of thyroid hormone receptors. The cDNAs coding for h c-erbA beta 1, alpha 1 and alpha 2 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-beta 62-81 immunoprecipitated only the beta 1 receptor. The antiserum anti-alpha 144-162 determined precipitation of both alpha 1 and alpha 2 proteins but not of the beta 1 receptor. Anti-alpha 2 431-451 produced a selective precipitation of alpha 2, and had no effect on alpha 1 or beta 1 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-beta 62-81 and anti-alpha 144-162 caused a partial shift of the labeled T3-receptor complex to a higher molecular form, while the antibody directed against c-erbA alpha 2 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.
Macchia
E
E
Istituto di Endocrinologia, Università di Pisa.
Falcone
M
M
Giorgilli
G
G
Bogazzi
F
F
Antonangeli
L
L
Baccarini
S
S
Fontanini
G
G
Torresani
J
J
DeGroot
L J
LJ
Pinchera
A
A
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D005333:Fetus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000737:chemistry*; D008297:Male; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D011988:Receptors, Thyroid Hormone / Q000032:analysis*
D000906:Antibodies; D011518:Proto-Oncogene Proteins; D011988:Receptors, Thyroid Hormone