Unidad de Fisiopatología Renal y Cardiovascular, Instituto Reina Sofía de Investigación Nefrológica, Universidad de Salamanca, Salamanca, Spain.
PLoS One. 2010 Jan 14;5(1):e8703. doi: 10.1371/journal.pone.0008703.
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.
Ras GTPases 被认为是细胞质蛋白，必须定位于细胞膜才能被激活，而在真核细胞的核内很少有任何 Ras 同工型存在的证据。
方法/主要发现：使用传统的抗体和免疫细胞化学、差速离心和 Western blot，我们观察到成纤维细胞和肾小球系膜细胞的核内存在 K-Ras 同工型的假定存在。为了避免与其他 Ras 同工型发生交叉反应，并使用针对 K-Ras（R-3400、H3845-M01、sc-30）或泛 Ras（05-516、OP40）的抗体在仅表达 K-Ras 同工型的细胞（从 H-ras(-/-)、N-ras(-/-)小鼠获得的成纤维细胞）中，我们也检测到一些核阳性表达。为了进一步探究核 K-Ras 的特性，我们通过交配 K-ras(+/-)杂合子小鼠生成了 K-Ras 敲除（K-ras(-/-)）胚胎成纤维细胞。使用特异性抗体，仅在 K-ras(-/-)成纤维细胞的细胞质中观察到 H-和 N-Ras 同工型。然而，通过免疫细胞化学和 Western blot，使用两种商业抗体（分别针对每种同工型的 sc-522 和 sc-521），在 K-ras(-/-)成纤维细胞的细胞质和核内均检测到 K-Ras4A 和 K-Ras4B 阳性信号。
结论/意义：我们表明，其他作者已经描述的成纤维细胞核内 K-Ras4B 的存在可能是由于抗体与未确定的核仁蛋白发生交叉反应所致。尽管这项研究还表明 K-Ras 同工型在成纤维细胞或肾小球系膜细胞中可能存在核表达，但它也揭示了在这些研究中，由于抗体的非特异性或细胞制剂的污染等重要限制，对于蛋白同工型的分布需要谨慎的重要性。
