English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/45709
logo share SHARE logo core CORE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:


Analysis of K-Ras nuclear expression in fibroblasts and mesangial cells

AuthorsFuentes-Calvo, Isabel; Santos de Dios, Eugenio ; López-Novoa, José M.
Issue Date14-Jan-2010
PublisherPublic Library of Science
CitationPLoS ONE 5(1): e8703
Abstract[Background]: Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells. [Methodology/Principal Findings]: Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras−/−,N-ras−/− mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras−/−) embrionary fibroblasts by mating of K-ras+/− heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras−/− fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras−/− fibroblasts. [Conclusions/Significance]: We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations.
DescriptionThis is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al
Publisher version (URL)http://dx.doi.org/10.1371/journal.pone.0008703
Appears in Collections:(IBMCC) Artículos
Files in This Item:
File Description SizeFormat 
10.1371-journal.pone.0008703.pdf384,77 kBAdobe PDFThumbnail
Show full item record
Review this work

Related articles:

WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.