Protein Isolation and Western Blotting

Protein samples (~1 mg) were isolated from the dorsal part of the thorax. Western blotting was carried out by using an SDS-containing gradient acrylamide gel, nitrocellulose blotting membrane, and TBST washing solution. In addition, 3% milk powder solved in TBST was used. Proteins were labeled at 4 °C overnight by the following primary antibodies: anti-ubiquitin (mouse, 1:500, Merck, Rahway, NJ, USA, ST1200), anti-Atg8a (rabbit, 1:2000 [64 (link),65 (link)]), anti-p62-vel (rabbit, 1:2000 [56 (link)]), anti-α-Tub84B (mouse, 1:2500, Sigma, St. Louis, MO, USA, T6199) and anti-EDTP (rat, 1:500 [19 (link)]). The following secondary antibodies were used (at room temperature for an hour): anti-rabbit IgG alkaline phosphatase (1:1000, Sigma, A3687), anti-mouse IgG alkaline phosphatase (1:1000, Sigma, A8438), and anti-rat IgG alkaline phosphatase (1:1000, Sigma, A8438). Primary and secondary antibodies were washed out 3 times for 10 min in TBST, and finally, membranes were incubated in an AP buffer. NBT/Bcip (Sigma, 72091) was used for recording the antibody staining, and NBT/Bcip was dissolved in an AP buffer (1:50).