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Correlation of GREB1 mRNA with protein expression in breast cancer: validation of a novel GREB1 monoclonal antibody

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Abstract

Studies of gene regulated by estrogen in breast cancer 1 (GREB1) have focused on mRNA levels with limited evidence about GREB1 protein expression in normal and breast cancer cells. A monoclonal antibody that recognizes GREB1 protein in breast tissues could be applied to correlate protein expression with established mRNA expression data. A hybridoma expressing a murine monoclonal antibody targeting a 119 amino acid peptide specific to human GREB1 was generated. The novel monoclonal GREB1 antibody (GREB1ab) was validated for use in Western blotting as well as immunohistochemical (IHC) applications. GREB1ab detects a 216 kDa protein corresponding to GREB1 in estrogen receptor alpha (ERα+) breast cancer cells as well as ERα− breast cancer cells transduced with a GREB1 expression vector. GREB1ab specificity was verified using an ERα antagonist to prevent GREB1 induction as well as a silencing siRNA targeting GREB1 mRNA. GREB1ab was further validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). ERα+ cell lines were observed to express GREB1 while ERα− cell lines did not express detectable levels of the protein. Using breast cancer tissue whole sections, IHC with the GREB1ab identified protein expression in ERα+ breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERα− breast cancer tissue. Furthermore, these data indicate that GREB1 mRNA expression correlates well with protein expression. The novel monoclonal GREB1ab is specific for GREB1 protein. This antibody will serve as a tool for investigations focused on the expression, distribution, and function of GREB1 in normal breast and breast cancer tissues.

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Acknowledgment

We gratefully acknowledge funding support in part by a grant from the Breast Cancer Research Foundation (Grant number N003173).

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Authors and Affiliations

  1. Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL, USA

    H. J. Hnatyszyn, M. Liu, L. Herbert, D. El-Ashry & M. E. Lippman

  2. Department of Pathology, Miller School of Medicine, University of Miami, Miami, FL, USA

    C. R. Gomez-Fernandez & M. Jorda

  3. Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

    A. Hilger, D. Thomas & J. M. Rae

Authors
  1. H. J. Hnatyszyn
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  2. M. Liu
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  3. A. Hilger
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  4. L. Herbert
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  5. C. R. Gomez-Fernandez
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  6. M. Jorda
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  7. D. Thomas
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  8. J. M. Rae
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  9. D. El-Ashry
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  10. M. E. Lippman
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Correspondence to H. J. Hnatyszyn.

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Hnatyszyn, H.J., Liu, M., Hilger, A. et al. Correlation of GREB1 mRNA with protein expression in breast cancer: validation of a novel GREB1 monoclonal antibody. Breast Cancer Res Treat 122, 371–380 (2010). https://doi.org/10.1007/s10549-009-0584-x

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  • DOI: https://doi.org/10.1007/s10549-009-0584-x

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