Including NEM in the lysis buffer allows for unambiguous detection of lipidated Atg8a and allowed the assessment of Atg8a and ref(2)P flux during prolonged starvation. 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. We found that the autophagic flux was highest during the initial 3 h of starvation, but diminished with continued starvation, and without reactivation of TORC1.
Siri Andresen, Amani Al Outa, Miriam Formica, Jorrit Enserink & Helene Knævelsrud (19 Jun 2025): Improved detection of lipidated Atg8a by immunoblotting in
Drosophila melanogaster cells and tissues enables precise investigation of Atg8a flux and its termination, Autophagy, DOI: 10.1080/15548627.2025.2508551
https://doi.org/10.1080/15548627.2025.2508551