Oligomeric Aβ42 was prepared as described before (71 (link)). Briefly, Aβ42 peptide powders were dissolved in hexafluoroisopropanol (HFIP) (Sigma-Aldrich, 52517) to remove preexisting aggregates. HFIP was evaporated in a fume hood overnight, followed by drying down in SpeedVac for 1 hour. The resulting peptide films were dissolved in dimethyl sulfoxide to prepare 5 mM stocks, sonicated in a bath sonicator for 10 min, and diluted to 100 μM in phenol-red free DMEM/F12 medium (Thermo Fisher Scientific, 21-041-025). To prepare Aβ oligomers, the stocks were incubated at 4°C for 24 hours. BV2 cells were seeded in a 24-well plate and transfected with Abi3-targeting or negative control siRNAs. Twenty-four hours after transfection, cells were treated with 200 nM oligomeric Aβ42 for 1 hour. The proteins were extracted using RIPA buffer, including protease and phosphatase inhibitors, and sonicated in a bath sonicator. The homogenates were centrifuged at 17,000g for 15 min and the supernatants were collected for Aβ measurement. Total protein amount was measured by bicinchoninic acid assay and intracellular Aβ levels were normalized by total protein amount for each sample.
Free full text: Click here
