Fürnrohr Barbara G, Mielenz Dirk
Division of Molecular immunology, Department of Internal Medicine III, Nikolaus-Fiebiger-Center, University of Erlangen-Nürnberg, Erlangen, Germany.
Methods Mol Biol. 2019;1929:595-605. doi: 10.1007/978-1-4939-9030-6_37.
Many Ca-binding proteins are differentially regulated under pro-inflammatory conditions in different organs. Specific quantification of RNA and protein expression of those proteins demands validated protocols. Peripheral blood mononuclear cells (PBMC) can mirror an inflammatory status originating from several organs and can therefore be an important diagnostic tool. Swiprosin-1/EFhd2 (EFhd2) is a ~30 kDa Ca and F-actin binding, cytoskeletal protein with two central EF hands and a C-terminal coiled-coil domain. Unbiased gene expression analyses and proteomics revealed that EFhd2 is regulated under pro-inflammatory conditions in several cell types and tissues. Here we describe validated protocols to quantify the expression of the human orthologue of Swiprosin-1/EFhd2 on RNA and protein level in PBMC. Both methods reveal that EFhd2 is stronger expressed in monocytes than in B cells of healthy donors. Thus, initial experiments relying on qPCR are likely to provide results with functional relevance. The higher expression of EFhd2 in monocytes could be related to monocyte migration under inflammatory conditions.
许多钙结合蛋白在不同器官的促炎条件下受到差异调节。对这些蛋白的RNA和蛋白质表达进行特异性定量需要经过验证的方案。外周血单核细胞（PBMC）可以反映源自多个器官的炎症状态，因此可以成为一种重要的诊断工具。Swiprosin-1/EFhd2（EFhd2）是一种约30 kDa的钙和F-肌动蛋白结合的细胞骨架蛋白，具有两个中央EF手型结构域和一个C端卷曲螺旋结构域。无偏倚的基因表达分析和蛋白质组学表明，EFhd2在几种细胞类型和组织的促炎条件下受到调节。在这里，我们描述了经过验证的方案，用于定量PBMC中Swiprosin-1/EFhd2人类同源物在RNA和蛋白质水平上的表达。两种方法都表明，在健康供体中，EFhd2在单核细胞中的表达比在B细胞中更强。因此，依赖定量PCR的初步实验可能会提供具有功能相关性的结果。EFhd2在单核细胞中的较高表达可能与炎症条件下的单核细胞迁移有关。
