Protein Expression Analysis in Drosophila Heads

Western blot samples were prepared from 10 female heads, which were treated in 32 μl of Fly Lysis buffer + 32 μl 2 × Laemmli buffer. 15 µl samples were run on 4–20% Mini-PROTEAN^® TGX™ Gel and blotted onto Nitrocellulose Membrane (Kisker Biotech, 40520100). After blocking with 3% Milk Powder (BioRad 170-6404 /Blotting-Grade Blocker/) dissolved in TBST, membranes were probed with specific antibodies [anti-Tubulin (1:1000, mouse, Sigma T6199), anti-Ref(2)P (1:2000, rabbit^{48 (link)}), anti-Atg8a (1:2500, rabbit^{50 (link)}), anti-EDTP, 1:1000, rat^{22 (link)}, anti-mouse IgG alkaline phosphatase (1:1000, Sigma, A8438), and anti-rabbit IgG alkaline phosphatase (1:1000, Sigma, A3687), anti-rat IgG alkaline phosphatase (1:1000, Sigma, A5153), and developed by NBT-BCIP solution (Sigma, 72091). Each western blot analysis was repeated at least three times with independent biological samples.