Improved detection of lipidated Atg8a by immunoblotting in Drosophila melanogaster cells and tissues enables precise investigation of Atg8a flux and its termination.

Macroautophagy/autophagy is an essential intracellular catabolic process for maintaining cellular homeostasis. In Drosophila melanogaster, Atg8a lipidation serves as a key marker for autophagy, yet traditional methods often fail to effectively detect its lipidated state. To overcome this limitation, we developed a refined approach that employs N-ethylmaleimide (NEM) to inhibit Atg4, thereby preserving Atg8a lipidation during sample preparation both in vitro and in vivo. We determined the optimal concentration of the autophagic inhibitors bafilomycin A1 (BafA1) and chloroquine (CQ) required for inhibition of autolysosomal degradation. Furthermore, we investigated the effects of prolonged nutrient deprivation on autophagic flux and TORC1 signaling. Our findings not only validate the effectiveness of this new approach to monitor lipidation of Atg8a but also provide insights into selection of autolysosomal inhibitors and nutrient-dependent regulatory roles of TORC1 in Drosophila.