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Multi-well Plate Immunoassays for Measuring Signaling Protein Activations/Deactivations and Membrane vs. Intracellular Receptor Levels

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Book cover Steroid Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1204))

Abstract

We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter signaling scenarios.

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References

  1. Bulayeva NN, Gametchu B, Watson CS (2004) Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways. Steroids 69:181–192

    Article  CAS  Google Scholar 

  2. Jeng YJ, Watson CS (2009) Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells: the involvement of rapidly activated kinases and caspases. BMC Cancer 9:334

    Article  Google Scholar 

  3. Vandenberg LN et al (2012) Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses. Endocr Rev 33:378–455

    Article  CAS  Google Scholar 

  4. Watson CS, Jeng YJ, Guptarak J (2011) Endocrine disruption via estrogen receptors that participate in nongenomic signaling pathways. J Steroid Biochem Mol Biol 127:44–50

    Article  CAS  Google Scholar 

  5. Campbell CH, Watson CS (2001) A comparison of membrane vs. intracellular estrogen receptor-alpha in GH(3)/B6 pituitary tumor cells using a quantitative plate immunoassay. Steroids 66:727–736

    Article  CAS  Google Scholar 

  6. Campbell CH, Bulayeva N, Brown DB, Gametchu B, Watson CS (2002) Regulation of the membrane estrogen receptor-alpha: role of cell density, serum, cell passage number, and estradiol. FASEB J 16:1917–1927

    Article  CAS  Google Scholar 

  7. Vinas R, Watson CS (2013) Rapid estrogenic signaling activities of modified (chlorinated, sulfonated, and glucuronidated) endocrine disruptor bisphenol A. Endocr Disruptors 1(1):e25411

    Article  Google Scholar 

  8. Anastasio NC, Witkin BM, Seitz PK, Watson CS, Cunningham KA (2009) Novel medium-throughput 96-well plate assay to immunohistochemically detect key brain proteins in the serotonin 5-HT2C receptor-ERK pathway. Society of Neuroscience Meeting, USA

    Google Scholar 

  9. Anastasio NC, Lanfranco MF, Bubar MJ, Seitz PK, Stutz SJ, McGinnis AG, Watson CS, Cunningham KA (2010) Serotonin 5-HT2C receptor protein expression is enriched in synaptosomal and post-synaptic compartments of rat cortex. J Neurochem 113:1504–1515

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Rajagopal S, Kim J, Ahn S, Craig S, Lam CM, Gerard NP, Gerard C, Lefkowitz RJ (2010) Beta-arrestin- but not G protein-mediated signaling by the “decoy” receptor CXCR7. Proc Natl Acad Sci U S A 107:628–632

    Article  Google Scholar 

  11. Watson CS, Jeng YJ, Hu G, Wozniak A, Bulayeva N, Guptarak J (2012) Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-alpha interactions with G protein-alphai and caveolin I. Steroids 77:424–432

    Article  CAS  Google Scholar 

  12. Pappas TC, Gametchu B, Yannariello-Brown J, Collins TJ, Watson CS (1994) Membrane estrogen receptors in GH3/B6 cells are associated with rapid estrogen-induced release of prolactin. Endocrine 2:813–822

    CAS  Google Scholar 

  13. Watson CS, Norfleet AM, Pappas TC, Gametchu B (1999) Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor. Steroids 64:5–13

    Article  CAS  Google Scholar 

  14. Campbell CH, Watson CS (2001) Regulation of the membrane estrogen receptor-a (mER-a) in the rat pituitary tumor cell line GH3/B6: the influence of cell density, passage number, serum starvation, and estradiol. Endocrine Society Meeting, Denver, CO

    Google Scholar 

  15. Gillies RJ, Didier N, Denton M (1986) Determination of cell number in monolayer cultures. Anal Biochem 159:109–113

    Article  CAS  Google Scholar 

  16. Lottering ML, Haag M, Seegers JC (1992) Effects of 17b-estradiol metabolites on cell cycle events in MCF-7 cells. Cancer Res 52:5926–5932

    CAS  PubMed  Google Scholar 

  17. Dzionek A, Fuchs A, Schmidt P, Cremer S, Zysk M, Miltenyi S, Buck DW, Schmitz J (2000) BDCA-2, BDCA-3 and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood. J Immunol 165:6037–6046

    Article  CAS  Google Scholar 

  18. Dzionek A, Sohma Y, Nagafune J et al (2001) BDCA-2, a novel plasmacytoid dendritic cell-specific type IIC-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction. J Exp Med 194:1823–1834

    Article  CAS  Google Scholar 

  19. Zivadinovic D, Watson CS (2005) Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells. Breast Cancer Res 7:R130–R144

    Article  CAS  Google Scholar 

  20. Zivadinovic D, Gametchu B, Watson CS (2005) Membrane estrogen receptor-alpha levels in MCF-7 breast cancer cells predict cAMP and proliferation responses PMCID: 15642158. Breast Cancer Res 7:R101–R112

    Article  CAS  Google Scholar 

  21. Watson CS, Alyea RA, Hawkins BE, Thomas ML, Cunningham KA, Jakubas AA (2006) Estradiol effects on the dopamine transporter: protein levels, subcellular location, and function. J Mol Signal 1:5

    Article  Google Scholar 

  22. Alyea RA, Laurence SE, Kim SH, Katzenellenbogen BS, Katzenellenbogen JA, Watson CS (2008) The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells. J Neurochem 106:1525–1533

    Article  CAS  Google Scholar 

  23. Alyea RA, Watson CS (2009) Nongenomic mechanisms of physiological estrogen-mediated dopamine efflux. BMC Neurosci 10:59

    Article  Google Scholar 

  24. Alyea RA, Watson CS (2009) Differential regulation of dopamine transporter function and location by low concentrations of environmental estrogens and 17beta-estradiol. Environ Health Perspect 117:778–783

    Article  CAS  Google Scholar 

  25. Seitz PK, Bremer NM, McGinnis AG, Cunningham KA, Watson CS (2012) Quantitative changes in intracellular calcium and extracellular-regulated kinase activation measured in parallel in CHO cells stably expressing serotonin (5-HT) 5-HT2A or 5-HT2C receptors. BMC Neurosci 13:25

    Article  CAS  Google Scholar 

  26. Vinas R, Watson CS (2013) Bisphenol S disrupts estradiol-induced nongenomic signaling in a rat pituitary cell line: effects on cell functions. Environ Health Perspect 121:352–358

    Article  Google Scholar 

  27. Vinas R, Watson CS (2013) Mixtures of xenoestrogens disrupt estradiol-induced nongenomic signaling and functions in pituitary cells. BMC Environ Health 12:26

    Article  CAS  Google Scholar 

  28. Jeng YJ, Kochukov MY, Watson CS (2009) Membrane estrogen receptor-alpha-mediated nongenomic actions of phytoestrogens in GH3/B6/F10 pituitary tumor cells. J Mol Signal 4:2

    Article  Google Scholar 

  29. Jeng YJ, Kochukov M, Watson CS (2010) Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells. Environ Health 9:61

    Article  Google Scholar 

  30. Watson CS, Jeng YJ, Kochukov MY (2008) Nongenomic actions of estradiol compared with estrone and estriol in pituitary tumor cell signaling and proliferation. FASEB J 22:3328–3336

    Article  CAS  Google Scholar 

  31. Jeng YJ, Watson CS (2011) Combinations of physiologic estrogens with xenoestrogens alter ERK phosphorylation profiles in rat pituitary cells. Environ Health Perspect 119:104–112

    Article  CAS  Google Scholar 

  32. Wozniak AL, Bulayeva NN, Watson CS (2005) Xenoestrogens at picomolar to nanomolar concentrations trigger membrane estrogen receptor-alpha-mediated Ca2+ fluxes and prolactin release in GH3/B6 pituitary tumor cells. Environ Health Perspect 113:431–439

    Article  CAS  Google Scholar 

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Acknowledgments

This work was jointly supported over the last 9 years by the National Institute of Environmental Health Sciences at the National Institutes of Health, the Department of Defense Breast Cancer Initiative, the Sealy Memorial Endowment Fund, the University of Texas Medical Branch Center for Addiction Research, the American Institute for Cancer Research, and the Passport Foundation, all in the USA.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 0645 312 Basic Science Building, Galveston, TX, 77555, USA

    Cheryl S. Watson, Yow-Jiun Jeng, Nataliya N. Bulayeva, Luke Y. Koong, Dragoslaca Zivadinovic & Rebecca A. Alyea

  2. Department of Surgery, Shriners Hospital for Children, University of Texas Medical Branch, Galveston, TX, USA

    Celeste C. Finnerty

  3. Department of Pediatrics, University of Texas Medical Branch, Galveston, TX, USA

    Terumi Midoro-Horiuti & Randall M. Goldblum

  4. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 0645 312 Basic Science Building, Galveston, TX, USA

    Terumi Midoro-Horiuti & Randall M. Goldblum

  5. Center for Addiction Research, University of Texas Medical Branch, Galveston, TX, USA

    Noelle C. Anastasio, Patricia K. Seitz & Thressa D. Smith

  6. Department of Pharmacology and Toxicology and Center for Addiction Research, University of Texas Medical Branch, Galveston, TX, USA

    Kathryn A. Cunningham

Authors
  1. Cheryl S. Watson
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  2. Yow-Jiun Jeng
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  3. Nataliya N. Bulayeva
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  4. Celeste C. Finnerty
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  5. Luke Y. Koong
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  6. Dragoslaca Zivadinovic
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  7. Rebecca A. Alyea
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  8. Terumi Midoro-Horiuti
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  9. Randall M. Goldblum
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  10. Noelle C. Anastasio
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  11. Kathryn A. Cunningham
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  12. Patricia K. Seitz
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  13. Thressa D. Smith
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Corresponding author

Correspondence to Cheryl S. Watson .

Editor information

Editors and Affiliations

  1. Second University of Naples, Naples, Italy

    Gabriella Castoria  & Ferdinando Auricchio  & 

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Watson, C.S. et al. (2014). Multi-well Plate Immunoassays for Measuring Signaling Protein Activations/Deactivations and Membrane vs. Intracellular Receptor Levels. In: Castoria, G., Auricchio, F. (eds) Steroid Receptors. Methods in Molecular Biology, vol 1204. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-1346-6_11

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  • DOI: https://doi.org/10.1007/978-1-4939-1346-6_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-1345-9

  • Online ISBN: 978-1-4939-1346-6

  • eBook Packages: Springer Protocols

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