Tla45

Hippocampal samples were homogenized with Tris-buffered saline (TBS) containing protease inhibitor cocktail (Roche Diagnostics Ltd, Mannheim, Germany) and phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The homogenates were ultracentrifuged at 32,000 x g, for 20 min, at 4 °C (Rotor: TLA45, Beckman Coulter). Protein concentrations of the supernatants and the sonicated pellets were determined using a BCA protein assay kit (Nacalai Tesque), and were adjusted to 1 ~ 2 μg/μL with sample buffer containing SDS. Samples were boiled at 96 °C for 10 min before being used for conventional SDS-PAGE, followed by transfer to nitrocellulose membranes. After blocking with skim milk (MEGMILK SNOW BRAND Co., Ltd., Tokyo, Japan), the membranes were incubated with the indicated primary antibody. After treatment with horseradish peroxidase-conjugated secondary antibody, the membranes were treated with reagent for exposure (Chemi-Lumi One Super, Nacalai Tesque; ImmunoStar LD, Wako, Osaka, Japan). Images of the membranes were captured with the ChemiDoc^TM XRS+ system (Bio-Rad, Hercules, CA) or a C-Digit Blot Scanner (LI-COR, Lincoln, NE), and analyzed using Image J software.

The HEV-LP purified by sucrose density gradient were mixed with 1.96 g of CsCl, and centrifuged at 35,000 rpm for 24 h at 4 °C in a Beckman SW50.1 rotor. The visible white band (at a density of 1.285 g/ml) was harvested by puncturing the tubes with a 21-gauge needle, diluted with EX-CELL 405 medium, and then centrifuged again in a Beckman TLA45 rotor at 45,000 rpm (125,000 × g) for 2 h to remove CsCl. The HEV-LP were placed on a carbon-coated grid, and the proteins were allowed to absorb into the grid for 5 min. After being rinsed with distilled water, the sample was stained with a 1% aqueous uranyl acetate solution and examined with a Hitachi H-7000 electron microscope (Hitachi High-Technologies, Tokyo, Japan). The chimeric HEV-LP were fixed on the grid, blocked with PBS containing 0.1% bovine serum albumin (PBS-BSA) at room temperature for 10 min, and then incubated with the c-myc- or HA-tagged antibody at room temperature for 15 min. After washing with PBS-BSA, the chimeric HEV-LP were incubated with gold-labeled second antibody at room temperature for 10 min and stained with 0.5% Uran for 5 sec after washing with PBS and distilled water.

APP_NL-H4 cells were plated at a density of 500,000 cells/well in 6-well plates. After resting for 24 h, the cells were treated with 1 ppm methanol extract of loquat leaves, UA or Amygdalin for 24 h, and the medium was replaced with the same medium containing the extract or amygdalin. The cells were incubated for 24 h and the medium was harvested. Aβ total of 42 levels in the medium from the APP_NL-H4 cells were measured using ELISA kits (IBL, Ltd., Fujioka, Japan).
Mouse cortices were homogenized with a tissue homogenizer (Rotor: TLA45, Beckman Coulter) on ice in Tris-buffered saline (TBS) (25 mM Tris–HCl pH 7.4, 137 mM NaCl, 2.68 mM KCl) containing a protease inhibitor cocktail (Roche Diagnostics Ltd, Mannheim, Germany) and phosphatase inhibitor cocktail (Nacalai Tesque). The homogenates were ultracentrifuged at 45,000×g for 20 min at 4 °C. The cortical Aβ42 levels were determined using a Human/Rat β amyloid (42) ELISA Kit (Wako, Tokyo, Japan). The results were measured using a Benchmark Microplate Reader (Bio-Rad, Hercules, CA, USA).