Kimura Shunsuke, Fujita Naonobu, Noda Takeshi, Yoshimori Tamotsu
Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Methods Enzymol. 2009;452:1-12. doi: 10.1016/S0076-6879(08)03601-X.
In this chapter, we introduce several methods that rely on the analysis of LC3, a versatile marker protein of autophagic structures in mammalian cultured cells. The appearance of LC3-positive puncta is indicative of the induction of autophagy, and it is observed either by immunofluorescence or by GFP-based microscopy. The maturation process by which autophagosomes are converted into autolysosomes can be monitored by the GFP and RFP tandemly tagged LC3 (tfLC3) method. Lysosomal turnover of LC3 is a good index of the proceeding of autophagy and can be assessed by Western blotting. These methods will provide a relatively easy assessment of autophagy, and the details of the procedure will be described along with possible pitfalls.
在本章中，我们介绍了几种依赖于对LC3进行分析的方法，LC3是哺乳动物培养细胞中自噬结构的一种多功能标记蛋白。LC3阳性斑点的出现表明自噬被诱导，可通过免疫荧光或基于绿色荧光蛋白（GFP）的显微镜观察到。自噬体转化为自溶酶体的成熟过程可通过绿色荧光蛋白和红色荧光蛋白串联标记的LC3（tfLC3）方法进行监测。LC3的溶酶体周转是自噬进程的一个良好指标，可通过蛋白质免疫印迹法进行评估。这些方法将提供相对容易的自噬评估，具体操作步骤细节将与可能出现的问题一并描述。
