Direct blue 71

Three whole flies per biological replicate were homogenized in boiling lysis buffer (50-mM Tris pH 6.8, 2% SDS, 10% glycerol, 100-mM dithiothreitol), sonicated, boiled for 10 minutes, and centrifuged at 13,300×g at room temperature for 10 minutes. Primary antibody used was rabbit polyclonal Anti-TBP (1:1,000, TFIID, Santa Cruz sc-421); Secondary antibody: peroxidase conjugated anti-Rabbit (1:5,000, Jackson Immunoresearch 115-155-003). Western blots were developed ChemiDoc (Bio-Rad) and quantified with ImageLab (Bio-Rad). For direct blue staining, PVDF membranes were submerged for 10 minutes in 0.008% Direct Blue 71 (Sigma-Aldrich) in 40% ethanol and 10% acetic acid, rinsed in 40% ethanol/10% acetic acid, air dried, and imaged. Western blots were performed using 5 biological replicates per genotype/sex, and Kruskal–Wallis test with Dunn's multiple comparisons was performed in GraphPad Prism.

Western blots were performed with either 3 whole adults or 5–10 flies collected during development, per biological sample (N), depending on the experiment and driver being used. Samples were homogenized in boiling fly lysis buffer (50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol (DTT)), briefly sonicated, boiled for 10 min and centrifuged for 8 min at 13,300× g at room temperature. PXi 4 (Syngene, Frederick, Maryland) or ChemiDoc (Bio-Rad) were used to develop the Western blots, which were then quantified with GeneSys (Syngene) or ImageLab (Bio-Rad), respectively. Quantification was conducted using the volume of whole lanes measuring ataxin-3 protein levels and corrected for its own background. Signal measured included the main ataxin-3 band and all other ataxin-3 species above it. The ataxin-3 signal from each lane was then divided by its own loading control (direct blue staining, whole lane signal measurement) and reported as arbitrary units. Direct blue stains of total protein were performed by submerging the PVDF membranes for 10 min in 0.008% Direct Blue 71 (Sigma-Aldrich, St. Louis, MO, USA) in 40% ethanol and 10% acetic acid and then rinsed with a solution of just 40% ethanol/10% acetic acid, before being air dried and imaged.

The staining solution of Direct blue 71 (Sigma-Aldrich, St. Louis, MO, USA) was prepared with 0.1% (w/v) in the double distilled water containing 40% ethanol, and 10% acetic acid. The nitrocellulose membrane with Aβ samples dotted was directly incubated in the staining solution and incubated for 10 min, then washed by double distilled water to acquire clear signals.

Direct Red 5B (DR5B), Direct Blue 71 (DB71), Reactive Black 5 (RB5), acid orange II (Sigma-Aldrich, Shanghai, China), hydrochloric acid, sodium hydroxide, sulphuric acid, chloroform, and CTAB (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) (Table S1) were all of analytical grade and diluted with purified distilled water according to the prescribed protocols of the manufacturers. The final concentration of the stock dye solutions was 1 g L⁻¹. The spent mushroom waste of Pleurotus ostreatus cultivation was provided by Huazhong Agricultural University (Wuhan, China).