Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method
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Author:
Date:
2017-05-20
[Abstract] RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic ...
[摘要] RNA代谢在不同的组织和发育阶段被严格控制,其失调是癌症的分子特征之一。 通过直接结合特定的序列元件,RNA结合蛋白(RBP)在共转录和转录后调控事件中起关键作用。 我们最近证实,在胰腺癌细胞中,获得耐药(DR) - 表型取决于多聚嘧啶区结合蛋白(PTBP1)的上调,其又被引入丙酮酸激酶前mRNA并有利于剪接 致癌性PKM2变体。 在这里,我们描述了紫外(UV)光交联和免疫沉淀(CLIP)方法的逐步方案,以确定RBP与贴壁人细胞系中其目标RNA的特定区域的直接结合。
背景 在细胞核中转录时,新生的RNA立即与被称为RNA结合蛋白(RBP)的反式因子立即组装。这些因子直接与RNA分子中特定的顺式调控序列相互作用,从而形成核糖核蛋白(RNP)复合物(Dreyfuss et al。,2002; ...
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Nuclei Isolation from Nematode Ascaris
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Author:
Date:
2017-05-05
[Abstract] Preparing nuclei is necessary in a variety of experimental paradigms to study nuclear processes. In this protocol, we describe a method for rapid preparation of large number of relatively pure nuclei from Ascaris embryos or tissues that are ready to be used for further experiments such as chromatin isolation and ChIP-seq, nuclear RNA analyses, or preparation of nuclear extracts (Kang et al., 2016; Wang et al., 2016).
[摘要] 在各种实验范例中准备核是必要的,以研究核过程。在本协议中,我们描述了一种从准备用于进一步实验(如染色质分离和ChIP-seq,核RNA)的蛔虫胚胎或组织快速制备大量相对纯的核的方法分析或准备核提取物(Kang等人,2016; Wang等人,2016)。
背景 核分离通常是研究核事件的分子和生物化学方面的第一步。已经开发了几种方法来分离来自不同组织和细胞类型的细胞核。然而,除了C以外的线虫的细胞核分离方案很少。已经描述了线索,(Ooi等人,2010; Zanin等人,2011; Haenni等人,2012, )。已经使用寄生线虫蛔虫的胚胎来制备用于体外无细胞系统的各种提取物(Cohen等人,2004) ; Lall等人,2004)。然而,这些提取物通常是全细胞提取物。在这里,我们描述了从线虫蛔虫制备细胞核的方法。
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