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Detection of variable levels of RARα and RARγ proteins in pluripotent and differentiating mouse embryonal carcinoma and mouse embryonic stem cells

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Abstract

Pluripotent mouse embryonal carcinoma (mEC) and mouse embryonic stem (mES) cells differentiate into several cell lineages upon retinoic acid (RA) addition. Differentiation is facilitated, in part, by RA activation of nuclear RA receptors (RARs) that bind to DNA response elements located in the promoters of target genes. The purpose of the studies reported here was to immunolocalize RARα and RARγ protein in mEC and mES cells and in their RA-induced differentiated progeny. Fixed cells were reacted with three different RARα antibodies and one RARγ antibody. Pluripotent and differentiated mEC and mES cells showed positive nuclear immunoreactivity with all antibodies tested. Two RARα antibodies also showed positive reactivity in the cytoplasm. Surprisingly, our results revealed variability in immunofluorescence intensity and in RARα and RARγ distribution from one cell to the other, suggesting that RARα and RARγ protein levels were not synchronous throughout the cell population. The results indicate that RARα and RARγ are present in pluripotent and differentiating mEC and mES cells and suggest that the expression of these proteins is dynamic.

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Acknowledgments

We thank James Asfour for technical support.

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Authors and Affiliations

  1. Tennessee State University, PO Box 1116, Room 313 Harned Hall, 3500 John A Merritt Boulevard, Nashville, TN 37209, USA

    FaMitah Q. Buchanan & Mary Ann Asson-Batres

  2. Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM/CNRS/Université de Strasbourg, 67404, Illkirch, France

    Cecile Rochette-Egly

Authors
  1. FaMitah Q. Buchanan
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  2. Cecile Rochette-Egly
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  3. Mary Ann Asson-Batres
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Correspondence to Mary Ann Asson-Batres.

Additional information

Funding from grant NIH/NIGMS/MBRS/SCORE S06 GM 008092 to M.A.A.B. was used to support these studies.

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Fig. S1

Western blot reacted with antibodies directed against retinoic acid receptor γ 453 (RARγ 453) and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nuclear (N) and cytosolic (C) extracts from P19 mEC (lanes 2, 3), BL6 mES (lanes 6, 7), and R1 mES (lanes 8, 9) cells were separated on the same gel and transferred to the same blot. The blot was probed with RARγ 453 and GAPDH antibodies. Molecular weights were determined from markers separated on the gel (lanes 1, 10) and transferred to the polyvinylidene difluoride membrane (lanes 4, 5 contained a sample not relevant to this study and were removed from the image). (JPEG 36 kb)

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Buchanan, F.Q., Rochette-Egly, C. & Asson-Batres, M.A. Detection of variable levels of RARα and RARγ proteins in pluripotent and differentiating mouse embryonal carcinoma and mouse embryonic stem cells. Cell Tissue Res 346, 43–51 (2011). https://doi.org/10.1007/s00441-011-1247-x

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  • DOI: https://doi.org/10.1007/s00441-011-1247-x

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