Fig 1. Overview of TR-FRET assay.

The TR-FRET signal relies on transfer of energy between two fluorophores, a donor and acceptor, when in close proximity. When two labeled molecules come together, excitation of the donor by an energy source triggers energy transfer towards the acceptor, which emits a fluorescent signal. Tb labeled LC3II donor and D2 labeled LC3II acceptor antibodies or Tb labeled donor p62 and Alexa-647 labeled acceptor p62 antibodies were used (A). Compounds that upregulate autophagy induce close proximity of the LC3II donor and acceptor antibodies, resulting in an accumulation of signal as AVs are formed; that signal is reduced over time as AVs are turned over. P62 donor and acceptor antibodies come in close proximity as they find p62 proteins, the signal declines over time as p62 is degraded (B). Compounds that block autophagy at the lysosome have a different signal. The LC3II signal accumulates as the donor and acceptor antibodies come together in new AVs, but there is no degradation of the signal since the AVs are not degraded. Similarly, there is no turnover of the p62 signal (C).