title: Analysis of RhoA and Rho GEF activity in whole cells and the cell nucleus.
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.
authors: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, North Carolina, USA.
authors: Christophe
authors: C
authors: Guilluy
authors: Adi D
authors: AD
authors: Dubash
authors: Rafael
authors: R
authors: García-Mata
mesh_terms: D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D064067:Rho Guanine Nucleotide Exchange Factors; D013347:Subcellular Fractions / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000032:analysis* / Q000378:metabolism
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein