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Author:
Date:
2014-03-05
[Abstract] Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize viral double-stranded RNA (dsRNA) which may be released into the extracellular milieu or spread from HCV-infected cells to uninfected neighbor cells via cell-to-cell contact, resulting in IFN-β activation that restricts viral propagation. We have found that MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. ...
[摘要] TLR3-Flag或Myc-MSR1与HCV RNA的免疫沉淀测定用于鉴定病毒RNA与识别病毒RNA以启动干扰素（IFN）信号传导（宿主细胞的关键抗病毒反应）的宿主蛋白的直接相互作用。 Toll样受体3（TLR3）和A类1型清道夫受体（MSR1）蛋白识别病毒双链RNA（dsRNA），其可以释放到细胞外环境或从HCV感染的细胞扩散到未感染的邻近细胞细胞间接触，导致限制病毒繁殖的IFN-β活化。我们已经发现MSR1结合细胞外dsRNA，介导其胞吞作用并向内吞体转运，其中TLR3参与其中，从而在感染和未感染的细胞中引发IFN应答。我们使用这个测定来证明MSR1在介导HCV RNA的TLR3识别中的关键作用。该方案中描述的测定基于具有条件缓冲液的常规蛋白质免疫沉淀方案，其防止裂解物中存在的RNA酶引起的非特异性RNA降解。与Flag-标记的蛋白相关的RNA分子被特异性抗体捕获，随后是蛋白G捕获，提取并通过RT-PCR测定定量检测，随后是用于可视化的琼脂糖凝胶电泳。这种方法也可以应用于其他蛋白质-RNA相互作用的检测。
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Author:
Date:
2013-09-20
[Abstract] Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) induces expression of both viral and cellular genes in virus infected B cells by mimicking activated Notch receptors (Notch-IC) that mediate transcription activation through binding to the repressing domain of the recombining binding protein suppressor of hairless (RBP-Jκ). In general, chromatin immunoprecipitation (ChIP) assays, electrophoresis mobility shift assays (EMSA), streptavidin-agarose mediated DNA pull-down assays, together with cell-based transcription reporter assays were conducted to verify whether the query protein is involved in EBNA2-dependent transcription. The ATP-bound state of nuclear chaperone nucleophosmin (NPM1) has been implicated in pleiotropic biological processes. An ATP-agarose-mediated pull-down protocol was ...
[摘要] EB病毒（EBV）核抗原2（EBNA2）通过模拟激活的Notch受体（Notch-IC）诱导病毒感染的B细胞中病毒和细胞基因的表达，所述Notch受体通过结合重组结合的抑制结构域介导转录激活无毛蛋白抑制剂（RBP-Jκ）。通常，进行染色质免疫沉淀（ChIP）测定，电泳迁移率变动测定（EMSA），链霉亲和素 - 琼脂糖介导的DNA下拉测定以及基于细胞的转录报道基因测定，以验证查询蛋白是否参与EBNA2依赖性转录。核伴侣核蛋白（NPM1）的ATP结合状态已涉及多效生物过程。开发ATP-琼脂糖介导的下拉方案以监测由ATP结合的NPM1诱导的引发前复合物的形成。根据EBNA2和Notch-IC已经显示出在B细胞系中靶基因的活化方面是部分可互换的，可以想象EBNA2是活化的Notch IC的生物学等价物。
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