Assessment of Posttranslational Modifications of ATG proteins.

Posttranslational modifications (PTMs) increase the functional diversity of proteins and play a key role in many cellular processes. Macroautophagy (hereafter simply referred to as autophagy) is an evolutionarily conserved, lysosome-dependent degradation pathway. This process is finely regulated by autophagy-related (ATG) genes widely conserved among eukaryotes from yeast to mammals. Various PTMs of ATG proteins such as phosphorylation, ubiquitination, and acetylation have been theorized to play a critical role in modulating autophagic processes and activity. In this chapter, we introduce several antibody-based tools (e.g., Western blot, Simple Western™, immunofluorescence, and immunoprecipitation) that are widely used to assess the PTMs of ATG proteins in mammalian cells.