LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells.

Autophagy is an intracellular degradation system that delivers cytoplasmic materials to the lysosome or vacuole. This system plays a crucial role in various physiological and pathological processes in living organisms ranging from yeast to mammals. Thus, an accurate and reliable measure of autophagic activity is necessary. However, autophagy involves dynamic and complicated processes that make it difficult to analyze. The term "autophagic flux" is used to denote overall autophagic degradation (i.e., delivery of autophagic cargo to the lysosome) rather than autophagosome formation. Immunoblot analysis of LC3 and p62/SQSTM1, among other proteins, has been widely used to monitor autophagic flux. Here, we describe basic protocols to measure the levels of endogenous LC3 and p62 by immunoblotting in cultured mammalian cells.