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Methods Enzymol. 2009;452:1-12. doi: 10.1016/S0076-6879(08)03601-X.

Monitoring autophagy in mammalian cultured cells through the dynamics of LC3.

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Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.


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.

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