| Detection of ASC Oligomerization by Western Blotting
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Author:
Date:
2017-05-20
[Abstract] The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1β. ASC oligomerization is direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1β. This protocol describes how to detect the oligomerization of ASC by Western blot.
[摘要] 具有半胱天冬酶募集区（ASC）衔接蛋白的凋亡相关斑点样蛋白桥联炎症体传感器和半胱天冬酶-1。在炎性体活化后，ASC以类似朊病毒的方式成核，成为负责募集和激活半胱天冬酶-1的大而单一的平台。活性胱天蛋白酶-1将反过来促进促炎细胞因子IL-1β的蛋白水解成熟。 ASC寡聚化是炎性体激活的直接证据，其检测允许读取与caspase-1和IL-1β无关。该方案描述了如何通过蛋白质印迹检测ASC的寡聚化。
背景 Inflammasomes是大量的多蛋白平台，其感测各种微生物，内源和环境胁迫因子，导致促炎IL-1细胞因子家族的成熟（Martinon等人，2002; Sharma和Kanneganti， 2016）。激活后，炎性细胞传感器通过pyrin结构域（PYD）-PYD同型相互作用募集衔接蛋白ASC。 ASC通过胱天蛋白酶激活和募集域（CARD）-CARD相互作用又结合半胱天冬酶-1，并有利于caspase-1的自我蛋白水解切割，导致IL-1β和IL-18的成熟（Hoss等人。，2016）。 Inflammasome激活引发ASC二聚体的超分子寡聚化成称为“ASC-specks”或“pyroptosome”（Fernandes-Alnemri等人，2007）的大交织原纤维。 ASC-speck / ...
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| Reconstruction of the Mouse Inflammasome System in HEK293T Cells
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Author:
Date:
2016-11-05
[Abstract] The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., 2016). When cells are exposed to microbes and/or danger signals, the inflammasome assembles and serves as a platform for the activation of caspase-1. Caspase-1 activation promotes the processing and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-33 as well as pyroptosis induction (Gross et al., 2011; Arend et al., 2008), which elicit inflammatory responses. Here, we describe how to co-transfect the ...
[摘要] 核糖体足迹或Ribo-seq，彻底改变了翻译研究。它最初是为培养中的酵母和哺乳动物细胞开发的（Ingolia等人，2009）。本文中，我们描述了来自先前公开的多核糖体分离程序的植物优化的亲手核糖体印迹方案（Ingolia等人，2009; Mustroph等人，2009 ）和核糖体印迹（Ingolia等人，2009; Ingolia等人，2013）。使用该协议，我们能够成功地分离和分析来自体外生长的拟南芥植物（黑暗生长的幼苗）的不同阶段的高质量核糖体印迹[Merchante < ，以及在液体培养物中生长的板和植物中的13天龄小植物[未发表的结果]）。
[背景] 翻译在调节基因活性中的中心作用早已被公认，但是在响应于特定刺激的全基因组翻译定量变化的系统探索最近才变得技术上可行。最初为培养中的酵母和哺乳动物细胞开发的核糖体印迹技术（通常称为Ribo-seq）已经彻底改变了翻译调节和基因表达的研究，因为其允许确定核糖体在基因组 - ...
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| Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria
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Author:
Date:
2015-01-20
[Abstract] Here we describe a protocol which we have used to study the homeostasis intracellular in vivo in lactic acid bacteria (LAB) using a fluorescent probe. This type of probes can be used for determining changes in the pH of cytoplasm with high sensitivity, temporal resolution and technical simplicity as well as accessing the rate of change of intracellular pH in response to a stimulus from kinetic measurements on short time scales (Breeuwer et al., 1996; Molenaar et al., 1991). This protocol has been designed to measure the intracellular pH using the pH-sensitive fluorescent probe 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) in LAB, Enterococcus faecalis (E. faecalis), Lactococcus lactis (L. lactis) and Lactobacillus ...
[摘要] 在这里我们描述了一个协议，我们已经用来研究乳酸细菌（LAB）使用荧光探针在体内细胞内体内平衡。 这种类型的探针可用于以高灵敏度，时间分辨率和技术简单性确定细胞质pH的变化，以及响应于来自短时间尺度上的动力学测量的刺激，获得细胞内pH的变化速率（Breeuwer& et al。，1996; Molenaar et al。，1991）。 该方案已经设计为使用pH敏感性荧光探针在LAB中的2'-，7'-双 - （2-羧乙基）-5（和-6） - 羧基荧光素（BCECF），粪肠球菌 （ E.cfaecalis ），乳酸乳球菌（<乳酸乳球菌）和干酪乳杆菌 em=""> L。casei ）。
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