Monitoring autophagy in mammalian cultured cells through the dynamics of LC3.
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.
Kimura
Shunsuke
S
Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Fujita
Naonobu
N
Noda
Takeshi
T
Yoshimori
Tamotsu
T
D001343:Autophagy; D002478:Cells, Cultured / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*
C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins