Quantification of Aβ and BACE-1 in Drosophila

Quantification of Aβ peptide and BACE-1 activity in fly brain were conducted as formerly detailed without modification [50 (link)]. In brief, for quantification of Aβ_1–42 peptide, 25 to 30 fly heads were homogenized in a 5 M guanidine-HCl containing protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at 12,000× g for 15 min at 4 °C. The supernatant was then subjected to human Aβ42 ELISA kit (Life Technologies, Invitrogen, Carlsbad, CA, USA). Concentration of Aβ was determined by comparing with standard Aβ_1–42. For BACE-1 activity, the same numbers of fly heads were homogenized in T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) without protease inhibitor. The lysate was then subjected to BACE-1 activity detection kit (Sigma-Aldrich, St. Louis, MO, USA). One unit of BACE-1 activity means it hydrolyzes 1.0 picomole of 7-Methoxycoumarin-4-acetyl-[Asn⁶⁷⁰, Leu⁶⁷¹]- Amyloid β/A4 Precursor Protein 770 Fragment 667-676- (2,4-dinitrophenyl)Lys-Arg-Arg amide substrate per minute at pH 4.5 at 37 °C.