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Analysis of RhoA and Rho GEF activity in whole cells and the cell nucleus

Nature Protocols volume 6, pages 2050–2060 (2011)Cite this article

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Abstract

We have recently shown that a fraction of the total cellular pool of the small GTPase RhoA resides in the nucleus, and that the nuclear guanine nucleotide exchange factor (GEF) Net1 has a role in the regulation of its activity. In this protocol, we describe a method to measure both the activities of the nuclear pools of RhoA and Rho GEFs. This process required the development of a nuclear isolation protocol that is both fast and virtually free of cytosolic and membrane contaminants, as well as a redesign of existing RhoA and Rho GEF activity assays so that they work in nuclear samples. This protocol can be also used for other Rho GTPases and Rho GEFs, which have also been found in the nucleus. Completion of the procedure, including nuclear isolation and RhoA or Rho GEF activity assay, takes 1 h 40 min. We also include details of how to perform a basic assay of whole-cell extracts.

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Figure 1: Characterization of nuclear isolation protocol.
Figure 2: Nuclear RhoA assay.
Figure 3: Nuclear Rho GEF assay.

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Acknowledgements

This work was supported by a Department of Defense Breast Cancer Predoctoral Fellowship (BC051092) to A.D.D., an American Heart Association Beginning Grant-in-Aid to R.G.-M. and a Marie Curie Outgoing International Fellowship from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 254747 to C.G. We thank K. Burridge for his continuous support, H. Mekeel for his assistance with electron microscopy and L. Sharek for her technical assistance.

AUTHOR CONTRIBUTIONS

R.G.-M. conceived and supervised the project, and devised and optimized the nuclear isolation strategy. R.G.-M., A.D.D. and C.G. devised and optimized the nuclear RhoA and Rho GEF activity pull-downs. R.G.-M. wrote the manuscript.

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Author notes

  1. Christophe Guilluy and Adi D Dubash: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Christophe Guilluy & Rafael García-Mata

  2. Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA

    Adi D Dubash

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  1. Christophe Guilluy
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Correspondence to Rafael García-Mata.

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Guilluy, C., Dubash, A. & García-Mata, R. Analysis of RhoA and Rho GEF activity in whole cells and the cell nucleus. Nat Protoc 6, 2050–2060 (2011). https://doi.org/10.1038/nprot.2011.411

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