Measurement of Autophagy in Cells and Tissues
Measurement of Autophagy in Cells and Tissues
复制标题
DOI：
10.1007/978-1-60761-756-3_13
复制
发表时间：
2010-01-01
期刊：
影响因子：
--
通讯作者：
Waguri, Satoshi
中科院分区：
文献类型：
--
作者：
Tanida, Isei;Waguri, Satoshi
两个主要的蛋白水解系统，泛素-蛋白酶体系统和自噬-溶酶体系统，有助于降解各种类型的蛋白质和/或蛋白质聚集体。一般来说，自噬-溶酶体系统参与蛋白质和细胞器的大量细胞内降解，而泛素-蛋白酶体系统是选择性的。在自噬过程中，胞质形式的LC 3（LC 3-I）与磷脂酰乙醇胺缀合以形成LC 3-磷脂酰乙醇胺缀合物（LC 3-II），其被募集到自噬体膜，并且LC 3-II在自噬体与溶酶体融合后被溶酶体水解酶降解。因此，LC 3-II的溶酶体周转反映了饥饿诱导的自噬活性，通过免疫印迹或免疫荧光检测LC 3已成为监测自噬的可靠方法。当自噬受损时，除了泛素化蛋白的积累之外，p62/SQSTM 1（泛素和LC 3结合蛋白）的水平也增加。在这里，我们描述的基本协议，分析内源性LC 3-II，p62，和自噬相关蛋白的免疫印迹，免疫荧光和电子显微镜。
Two major proteolysis systems, the ubiquitin-proteasome system, and the autophagy-lysosome system, contribute to degradation of various types of protein and/or protein aggregates. In general, the autophagy-lysosome system is involved in bulk intracellular degradation of proteins and organelles, while the ubiquitin-protcasome system is selective. During autophagy, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes, and LC3-II is degraded by lysosomal hydrolases after the fusion of autophagosomes with lysosomes. Therefore, lysosomal turnover of LC3-II reflects starvation-induced autophagic activity, and detection of LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy. When autophagy is impaired, the level of p62/SQSTM 1, a ubiquitin- and LC3-binding protein, is increased in addition to the accumulation of ubiquitinated proteins. Here, we describe basic protocols to analyze endogenous LC3-II, p62, and autophagyrelated proteins by immunoblotting, immunofluorescence, and electron microscopy.