Fractionation of Drosophila and Human Samples

Fractionations of both overexpression and CRISPR Drosophila models were conducted as previously described [14 (link)]. In brief, 25 third instar larvae (for the overexpression models) or 1–2 days old 25 homozygous flies (for the CRISPR lines) were homogenized in Trizol (Thermofisher Scientific 15596026). Lysates were then centrifuged at 25,000×g for 30 min. The supernatant became the soluble fraction and the pellet was solubilized in urea buffer (30 mM Tris, 7 M Urea, 2 M Thiourea, 4% CHAPS, 1X Protease Inhibitor Cocktail (Millipore Sigma 11873580001), 0.5 mM PMSF, RNAsin Plus 400 units/ml (Fischer Scientific PRN2615), pH 8.5) to generate the urea/insoluble fraction. For human fractionation samples, post-mortem tissue (four spinal cord and two cerebellum samples as controls) was homogenized and subjected to the fractionation protocol described above.