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Puromycin dihydrochloride

嘌呤霉素

公司名称: Sigma-Aldrich
产品编号: P9620
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Analysis of Exosome Transfer in Mammalian Cells by Fluorescence Recovery after Photobleaching
Author:
Date:
2018-01-20
[Abstract]  During the course of evolution, prokaryote and eukaryote cells have developed elegant and to some extent analogous strategies to communicate with each other and to adapt to their surrounding environment. Eukaryotic cells communicate with each other through direct interaction via juxtracrine signaling and/or by secreting soluble factors. These secreted factors can subsequently act on the cell itself (autocrine signaling) or interact with neighboring (paracrine signaling) and distant (endocrine signaling) cells. The transmission of signals between cells and tissues has been traditionally ... [摘要]  在进化过程中,原核生物和真核生物细胞已经形成了优雅,在一定程度上类似的相互交流和适应周围环境的策略。真核生物细胞通过直接相互作用经Juxtracrine信号传导和/或通过分泌可溶性因子相互沟通。这些分泌的因子随后可以作用于细胞本身(自分泌信号传导)或与邻近的(旁分泌信号传导)和远处的(内分泌信号传导)细胞相互作用。传统上认为细胞和组织之间的信号传递受到基于蛋白质的信号传导系统的调节。通常,通过胞吐作用分泌到细胞外环境中的蛋白质含有典型的分泌靶向序列(Théry等,2002)。然而,具有非连续和刺激依赖性分泌的蛋白质,不含有经典分泌靶向序列的蛋白质,以及在细胞外环境(DNA,mRNA,肽,代谢物,miRNA和其他RNA)中可能过于不稳定的物质物种)可以以特定的方式分泌在小膜胞外囊泡(EV)中(Hagiwara等人,2014)。外来体代表直径为30-130nm的这些分泌的膜囊泡中的一大类(Cocucci等人,2009;Théry等人,2009; Kowal等人, >,et al。,2014),其形成在称为多泡体的内体隔室中的分泌细胞内。加载到外泌体中的分子以及细胞之间外来体转移的强度是随后调节受体细胞的重要参数。目前关于外泌体分泌及其在受体细胞中内化的知识仍不完整。已知外泌体的分泌强度根据细胞类型和其生理状态而变化(Garcia等人,2016)。此外,促进与细胞 - ...

An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins
Author:
Date:
2017-11-20
[Abstract]  We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein ... [摘要]  我们最近报道了一种针对内源性感兴趣蛋白(POI)的靶向蛋白水解的亲和指导PROtein导弹(AdPROM)系统(Fulcher等人,2016和2017)。 AdPROM由Von Hippel Lindau(VHL)蛋白组成,Cullin 2 E3连接酶底物受体(Bosu and Kipreos,2008),与识别细胞中靶蛋白的高亲和力多肽结合剂(如骆驼科纳米抗体)缀合。当在细胞中引入时,靶蛋白质被招募到CUL2 E3泛素连接酶复合体用于泛素介导的蛋白酶体降解。对于靶蛋白的募集,我们使用了基于人类III型纤连蛋白结构域的骆驼科动物来源的VHH结构域纳米抗体以及合成多肽单体(Sharm等人,2013; Fridy等人。,2014; Schmidt et al。,2016)。在此协议中,我们描述了生成AdPROM构建体及其在人细胞系中用于靶蛋白质破坏的详细方法。 AdPROM允许对POI进行功能表征,并且其目标蛋白质破坏的效率克服了RNA干扰方法的许多局限性,这些方法需要长时间的治疗并与脱靶效应相关联,而CRISPR / Cas9基因编辑并不总是可行的。
【背景】该协议使人们能够在哺乳动物细胞系中设计,构建和表达AdPROM VHL-nano ...

Isolation and Immortalization of Fibroblasts from Different Tumoral Stages
Author:
Date:
2014-04-05
[Abstract]  Tumour microenvironment and cancer-associated fibroblasts in particular exhibit tumour promoting abilities that are not present in their normal counterparts (Calvo et al., 2013; Hanahan and Coussens, 2012). Therefore, functional and molecular characterization of the modifications occurring in fibroblasts during tumour progression is essential to fully understand their role in tumour progression. Previous studies have addressed this issue using human fibroblasts and comparing normal and adjacent fibroblasts to tumour-associated fibroblasts (Kalluri and Zeisberg, 2006). However, ... [摘要]  肿瘤微环境和癌相关成纤维细胞特别表现出其正常对应物中不存在的肿瘤促进能力(Calvo等人,2013; Hanahan和Coussens,2012)。因此,在肿瘤进展期间发生在成纤维细胞中的修饰的功能和分子表征是完全理解它们在肿瘤进展中的作用所必需的。以前的研究已经解决了这个问题使用人类成纤维细胞和比较正常和相邻成纤维细胞与肿瘤相关的成纤维细胞(Kalluri和Zeisberg,2006)。然而,这些研究受到人类样品的内在变异性(例如配对,年龄,基因组景观,等)的阻碍。为了克服这些问题,我们使用了完全表征的小鼠乳腺癌模型MMTV-PyMT(Guy等人,1992; Lin等人,2003)。 MMTV-PyMT转基因小鼠在小鼠乳腺肿瘤病毒启动子/增强子的指导下表达多瘤病毒中T抗原。这是一个多焦点管腔乳腺癌模型,经历良好定义和特征阶段(即增生,腺瘤,癌和浸润性癌)。有趣的是,这种模型有100%的发病率,是非常结构性的(呈现高浓度的成纤维细胞),并引起肺自发转移80-94%的发病率。重要的是,至少对于腹股沟乳腺(腺4和9),不同的肿瘤阶段与小鼠的年龄很好相关:在6周龄时出现的增生,6-8周龄之间的腺瘤,癌和侵袭性癌症从8周开始。该模型使我们可靠地分离来自不同肿瘤阶段的成纤维细胞,并仔细表征其功能和分子性质(Calvo等人,2013)。

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