A biochemical reconstitution approach to identify autophagy receptors for aggrephagy in mammalian cells.

Aggrephagy is a major way to clear protein aggregates. Here, we describe a pipeline of experiments to find autophagy receptors for aggrephagy. Steps include an in vitro reconstitution to recapitulate autophagosome recognizing aggregates and receptor identification steps based on flow cytometry and mass spectrometry. We also describe functional validation steps based on immunofluorescence and immunoblot. The protocol provides a practical way to identify aggrephagy receptors. For complete details on the use and execution of this protocol, please refer to Ma et al. (2022).