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Detection of retinoid X receptors using specific monoclonal and polyclonal antibodies
C Rochette-Egly1, Y Lutz, V Pfister
1Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine 11, Strasbourg, France.
Abstract:
Because of the growing importance of the Retinoid X Receptors (RXR alpha, beta and gamma) in the retinoid acid signalling pathway, we have prepared polyclonal and monoclonal antibodies directed against these proteins. For this purpose, either the whole mouse RXR alpha protein expressed in E.Coli, or synthetic peptides corresponding to amino acid sequences common to all RXRs or unique to RXR alpha, beta or gamma, were used as antigens. Antibodies recognizing either all three RXR types (alpha, beta and gamma) or specific for each RXR type were obtained. The antibodies were characterized by immunocytochemistry, immunoblotting, immunoprecipitation and electromobility shift assay (EMSA). These antibodies allowed us to detect the presence of RXR alpha proteins in mouse embryos and in mouse embryonal carcinoma cells (F9 and P19 cell lines) by immunoblotting, immunoprecipitation and EMSA whereas RXR beta could be detected only by EMSA and RXR gamma could not be detected by any of these techniques.
Insights
Researchers developed antibodies for Retinoid X Receptors (RXRs). These tools detect RXR alpha and beta in cells and embryos, but not RXR gamma, advancing retinoid signaling pathway research.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Retinoid X Receptors (RXRs) are crucial nuclear receptors in retinoid acid signaling.
- Understanding RXR subtypes (alpha, beta, gamma) is vital for deciphering their distinct biological roles.
- Development of specific antibodies is essential for studying RXR expression and function.
Purpose of the Study:
- To generate and characterize polyclonal and monoclonal antibodies against Retinoid X Receptor (RXR) alpha, beta, and gamma.
- To validate the specificity and utility of these antibodies in detecting RXRs in biological samples.
- To investigate the expression patterns of RXR subtypes in mouse embryos and cell lines.
Main Methods:
- Production of antibodies using recombinant mouse RXR alpha protein and synthetic peptides common or unique to RXR subtypes as antigens.
- Characterization of antibody specificity and function through immunocytochemistry, immunoblotting, immunoprecipitation, and electromobility shift assay (EMSA).
- Application of validated antibodies to detect RXR expression in mouse embryos and F9/P19 embryonal carcinoma cell lines.
Main Results:
- Successfully generated antibodies recognizing all three RXR subtypes (alpha, beta, gamma) or specific to individual subtypes.
- Detected RXR alpha in mouse embryos and cell lines via immunoblotting, immunoprecipitation, and EMSA.
- Detected RXR beta exclusively by EMSA, and RXR gamma was not detectable by any tested method.
- Antibody characterization confirmed their specificity and effectiveness in various assays.
Conclusions:
- Developed a valuable toolkit of antibodies for studying RXR alpha and beta.
- Demonstrated differential expression and detectability of RXR subtypes in early mouse development and cell lines.
- Highlighted the need for further investigation into RXR gamma detection and function.