Protein Lipidation - Methods and Protocols
Protein Lipidation - Methods and Protocols
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蛋白质脂化 - 方法和方案
DOI：
10.1007/978-1-4939-9532-5_12
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
McClafferty H
中科院分区：
文献类型：
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作者：
McClafferty H
缺乏特定的药理学工具来询问棕榈酰酰基转移酶（zDHHCs）在哺乳动物细胞中的功能作用，显着阻碍了对这个重要基因家族的理解。通过RNA干扰（RNAi）的基因沉默是真核生物中允许通过靶向其编码mRNA来特异性敲低蛋白质表达的过程。因此，RNAi可以用作蛋白质组学工具，通过分析内源性zDHHC敲低对其蛋白质靶标或途径的影响来研究特定zDHHC在细胞中的功能作用。在这里，我们描述了应用短干扰RNA（siRNA），一类短（20-25个碱基对）双链RNA，敲低人胚肾（HEK 293）细胞中表达的内源性zDHHC酶，随后使用RT-qPCR定量zDHHC mRNA水平验证敲低效率。
The lack of specific pharmacological tools to interrogate the functional role of palmitoyl acyltransferases (zDHHCs) in mammalian cells has significantly hampered the understanding of this important gene family. Gene silencing by RNA interference (RNAi) is a process in eukaryotes that allows specific knockdown of the expression of proteins by targeting their coding mRNA. RNAi can thus be used as a proteomic tool to study the functional role of specific zDHHCs in cells by analyzing the effects of endogenous zDHHC knockdown on their protein targets or pathways. Here we describe the application of short interfering RNA (siRNA), a class of short (20–25 base pairs) double-stranded RNAs, to knockdown endogenous zDHHC enzymes expressed in human embryonic kidney (HEK293) cells and subsequent validation of knockdown efficiency using RT-qPCR to quantify zDHHC mRNA levels.