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Domain-Specific Monoclonal Antibodies Against Human Rev-erbβ

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Abstract

The nuclear receptor Rev-erbβ is a potent transcriptional factor whose functional study has been limited by the lack of suitable antibodies against it. To better understand Rev-erbβ’s biological roles, we generated five hybridoma cell lines secreting antibodies against human Rev-erbβ in mice immunized with the purified, prokaryotically expressed recombinant Rev-erbβ-6His fusion protein. Using Western blotting and immunofluorescence analyses, all the five monoclonal antibodies (MAbs) showed strong immunoreactivity to both prokaryotically and eukaryotically expressed recombinant Rev-erbβ. An immunoprecipitation study showed that all five monoclonal antibodies against Rev-erbβ were able to pull down the recombinant Rev-erbβ-Flag protein, but only one of the MAbs against Rev-erbβ, 37H8, could pull down the endogenous Rev-erbβ protein. Furthermore, domain specificity of these MAbs was characterized. Due to the high similarities between Rev-erbα and Rev-erbβ in the C and E domains, those C and E domain-specific anti-Rev-erbβ antibodies can react with human Rev-erbα as well. The MAbs produced in the study will provide a valuable tool for investigating the function of Rev-erbβ.

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Acknowledgements

We are thankful to Yaling Zhang for the technical assistance. This work was supported by the Fundamental Research Funds for the Central Universities (GK201504009), research grants to H.X. from the National Natural Science Foundation of China (No. 81272543, No. 81471772), the Natural Science Foundation of Shaanxi Province, China (No. 2014JM4113) and the Science and Technology Department of Shaanxi province (2012K19-02-03).

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Authors and Affiliations

  1. Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang’an Road, Xi’an, 710062, Shaanxi, People’s Republic of China

    Fang Chen, Yanqing Li, Junli Zhao & Haibin Xia

  2. School of Modern Agriculture and Biotechnology, Ankang University, Ankang, 725000, Shaanxi, People’s Republic of China

    Fang Chen

  3. Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA

    Qinwen Mao

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  1. Fang Chen
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  2. Yanqing Li
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  3. Junli Zhao
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  4. Qinwen Mao
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  5. Haibin Xia
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Correspondence to Haibin Xia.

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All of the animal care and use procedures were approved by the guidelines of the Institutional Animal Care and Use Committee of Shaanxi Normal University.

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The authors declare that they have no conflict of interest.

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Chen, F., Li, Y., Zhao, J. et al. Domain-Specific Monoclonal Antibodies Against Human Rev-erbβ. Appl Biochem Biotechnol 182, 978–989 (2017). https://doi.org/10.1007/s12010-016-2375-2

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  • DOI: https://doi.org/10.1007/s12010-016-2375-2

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