| Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy
|
|
Author:
Date:
2018-06-20
[Abstract] Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition receptors (PRRs) capable of detecting viral dsRNAs such as endosomal toll-like receptor 3 (TLR3) and cytosolic RIG-I-like receptors (RLRs) which lead to the production of type I interferons (IFNs). Thus, intracellular localization of viral dsRNA can provide insight into the downstream signaling pathways leading to innate immune activation. Here, we describe a quantitative method for measuring extracellular dsRNA uptake and visualizing subcellular ...
[摘要] 双链RNA是一种有效的病原体相关分子模式（PAMP），作为病毒复制的副产物和病毒感染的众所周知的标志产生。 病毒dsRNA可以从感染的细胞释放到细胞外空间并通过胞吞作用被邻近细胞内化。 哺乳动物具有能够检测导致产生I型干扰素（IFN）的病毒dsRNA（例如内体Toll样受体3（TLR3）和胞质RIG-1样受体（RLR））的多模式识别受体（PRR）。 因此，病毒dsRNA的细胞内定位可以提供对导致先天免疫激活的下游信号传导途径的了解。 在这里，我们描述了一种测量细胞外dsRNA摄取和分别通过流式细胞仪和共聚焦显微镜观察内化dsRNA的亚细胞定位的定量方法。
【背景】双链RNA（dsRNA）是病毒复制的常见副产物，通过产生I型干扰素（IFN）和其他促炎细胞因子（Nellimarla和Mossman，2014）是抗病毒免疫的有效激活剂。病毒的dsRNA通过TLR3核内体内所感测（松本等人，2003年）或在由RIG-I样受体（RLRS），RIG-I和MDA-5（加藤等人，2006）。在裂解感染，这些dsRNA可以被释放到细胞外空间，它们结合于相邻小区，如A类清道夫受体（SR-A）和RAFTLIN表面受体，并且随后经由网格蛋白介导的内吞作用内在化（伊藤制 2008; DeWitte-Orr等人，2010; Watanabe等人，2011; Dansako等人，， ...
|
|
|
| UV Cross-linking Assay and Competition Assay
|
|
Author:
Date:
2012-12-20
[Abstract] UV cross-linking assay is a standard method used to detect protein-RNA interaction. This method takes advantage of UV irradiation to trigger the formation of the covalently bonded RNP (ribonucleoprotein) complex that is more stable and makes it possible to be isolated in the denaturing conditions. Briefly, 32P-labeled RNA probe and proteins are incubated to form RNP complexes spontaneously; the mixture is then exposed to UV irradiation, followed by treatment with ribonuclease to remove RNA fragments not covalently bound to protein. The oligoribonucleotide-protein complexes are analyzed by SDS-PAGE, and the signals visualized by phosphorimaging. Competitive UV cross-linking assay is a method to determine the protein binding sites and specificity on the RNA substrate. In this ...
[摘要] UV交联测定是用于检测蛋白-RNA相互作用的标准方法。该方法利用UV辐射来触发更稳定的共价结合的RNP（核糖核蛋白）复合物的形成，并且使得可以在变性条件下分离。简言之，将32 P标记的RNA探针和蛋白质自发孵育以形成RNP复合物;然后将混合物暴露于UV辐射，随后用核糖核酸酶处理以除去未与蛋白质共价结合的RNA片段。通过SDS-PAGE分析寡核糖核苷酸 - 蛋白复合物，并通过磷光成像显现信号。竞争性UV交联测定是确定蛋白质结合位点和对RNA底物的特异性的方法。在该测定中，将过量的未标记的竞争剂RNA与蛋白质预孵育，然后加入32 P标记的RNA探针。如果竞争剂RNA包含蛋白质结合区，则蛋白质和RNA探针之间的结合将竞争并且放射性结合信号将降低。
|
|
|