| Fluorescent Polysome Profiling in Caenorhabditis elegans
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Author:
Date:
2020-09-05
[Abstract] An important but often overlooked aspect of gene regulation occurs at the level of protein translation. Many genes are regulated not only by transcription but by their propensity to be recruited to actively translating ribosomes (polysomes). Polysome profiling allows for the separation of unbound 40S and 60S subunits, 80S monosomes, and actively translating mRNA bound by two or more ribosomes. Thus, this technique allows for actively translated mRNA to be isolated. Transcript abundance can then be compared between actively translated mRNA and all mRNA present in a sample to identify instances of post-transcriptional regulation. Additionally, polysome profiling can be used as a readout of global translation rates by quantifying the proportion of actively translating ribosomes within a ...
[摘要] [摘要 ] 基因调控的一个重要但经常被忽视的方面发生在蛋白质翻译的水平。许多基因不仅受转录调控，还受其被募集以主动翻译核糖体（多核糖体）的倾向性的调节。多核糖体分析允许分离未结合的40S和60S亚基，80S单核糖体，并主动翻译由两个或多个核糖体结合的mRNA。因此，该技术允许分离出主动翻译的mRNA。然后可以在主动翻译的mRNA和样品中存在的所有mRNA之间比较转录本丰度，以鉴定转录后调控的实例。此外，多核糖体分析可以用作ar 通过量化样品中活性翻译核糖体的比例来确定总体翻译率。先前建立的多核糖体谱分析方案依赖于RNA的吸收来可视化级分中多核糖体的存在。但是，随着能够检测荧光的流通池的出现，除了RNA的吸收以外，还可以检测和定量荧光标记的蛋白质与多核糖体的结合。该协议提供了有关如何在秀丽隐杆线虫中进行荧光多核糖体分析以收集主动翻译的mRNA，定量整体翻译的变化以及检测核糖体结合伴侣的详细说明。
[背景 ] 绑定到同一个mRNA转录多积极核糖体的翻译被称为多聚核糖体。可使用多核糖体分析将多核糖体与其他核糖体形式和未结合的mRNA分离。P olysome 分析已经在蛋白质翻译领域的关键技术。与mRNA的-SEQ的同时，多边形OME分析允许由转录后机制调节以进行检测和定量转录物（兰等人，2019;罗林。等人，2019） ，通过定量PCR （熊猫等人， ...
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| Immunohistochemical Identification of Human Skeletal Muscle Macrophages
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Author:
Kate Kosmac, Bailey D. Peck, R. Grace Walton, Jyothi Mula, Philip A. Kern, Marcas M. Bamman, Richard A. Dennis, Cale A. Jacobs, Christian Lattermann, Darren L. Johnson and Charlotte A. Peterson,
Date:
2018-06-20
[Abstract] Macrophages have well-characterized roles in skeletal muscle repair and regeneration. Relatively little is known regarding the role of resident macrophages in skeletal muscle homeostasis, extracellular matrix remodeling, growth, metabolism and adaptation to various stimuli including exercise and training. Despite speculation into macrophage contributions during these processes, studies characterizing macrophages in non-injured muscle are limited and methods used to identify macrophages vary. A standardized method for the identification of human resident skeletal muscle macrophages will aide in the characterization of these immune cells and allow for the comparison of results across studies. Here, we present an immunohistochemistry (IHC) protocol, validated by flow cytometry, to distinctly ...
[摘要] 巨噬细胞在骨骼肌修复和再生中具有很好的特征。关于驻留巨噬细胞在骨骼肌动态平衡，细胞外基质重塑，生长，代谢和适应各种刺激（包括运动和训练）中的作用知之甚少。尽管在这些过程中推测了巨噬细胞的贡献，但表征非受伤肌肉中的巨噬细胞的研究是有限的，用于鉴定巨噬细胞的方法各不相同。用于鉴定人类骨骼肌巨噬细胞的标准化方法将有助于鉴定这些免疫细胞，并可用于各研究结果的比较。在这里，我们提出免疫组织化学（IHC）协议，通过流式细胞术验证，以清楚地识别常驻人类骨骼肌巨噬细胞种群。我们显示CD11b和CD206双IHC有效识别人骨骼肌中的巨噬细胞。此外，非受伤人骨骼肌中的大多数巨噬细胞表现出“混合”M1 / M2表型，表达CD11b，CD14，CD68，CD86和CD206。在休息的骨骼肌中存在相对较少的CD11b + / CD206-巨噬细胞群。这种人口的相对丰度的变化可能反映了骨骼肌肉环境的重要变化。肌肉中的CD11b和CD206 IHC也显示出巨噬细胞的不同形态学特征，这些特征可能与这些细胞的功能状态有关。
【背景】巨噬细胞是能够适应局部微环境变化的多向性免疫细胞。在过去几年中，研究表明巨噬细胞表型是动态的，存在于连续统一体中（Mosser and Edwards，2008，Italiani and Boraschi，2014，Martinez and ...
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| Measurement of TACE Activity in Extracts from Cultured Cells
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Author:
Date:
2014-10-20
[Abstract] In cigarette smoke–induced and inflammation-associated lung cancer development, cigarette smoke extract (CSE) activates tumor necrosis factor-alpha (TNF-α) secretion from macrophages. TNF-α converting enzyme (TACE), also known as α-Secretase or ADAM17 (A Disintegrin and Metalloprotease), is a member of the ADAM family of metalloproteases. TACE mediated ectodomain shedding leads to the conversion of the inactive TNF-α precursor into the active mature pro-inflammatory cytokine. The SensoLyte 520 TACE (α-Secretase) Activity Assay Kit was used to detect TACE activity in CSE-activated macrophages. This assay is reliable, reproducible and easy to carry out in 96 well plate format.
[摘要] 在香烟烟雾诱导和炎症相关的肺癌发展中，香烟烟雾提取物（CSE）激活巨噬细胞的肿瘤坏死因子-α（TNF-α）分泌。 TNF-α转化酶（TACE），也称为α-分泌酶或ADAM17（解聚素和金属蛋白酶），是金属蛋白酶的ADAM家族的成员。 TACE介导的胞外结构域脱落导致无活性的TNF-α前体转化为活性成熟促炎细胞因子。 SensoLyte 520 TACE（α-分泌酶）活性测定试剂盒用于检测CSE活化的巨噬细胞中的TACE活性。 该测定法是可靠的，可重复的并且容易在96孔板形式中进行。
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