Anti ece 1

Total protein concentration of the brain was determined using the Bradford protein assay (Bio-Rad; Hercules, CA, USA). Brain proteins diluted to 1 μg/μL were treated with 4× LDS sample buffer (Invitrogen; Carlsbad, CA, USA) and 5 mM dithiothreitol before heating at 70 °C for 10 min. The denatured sample solution was subjected to Western blotting, following SDS-PAGE on a 10% Bis-Tris gel (Invitrogen) and subsequent transfer to PVDF (0.22 μm pore size, Bio-Rad). PVDF membranes were blocked in 2.5% ECL prime blocking (GE Healthcare; Madison, WI, USA) dissolved in phosphate-buffered saline (PBS) containing 0.5% Tween-20 (PBS-T), and incubated with primary antibody at the following dilutions: 1:1000 anti-Aβ (4G8) (Signet; Dedham, MA, USA), 1:1000 anti-APP(N) (IBL), 1:1000 anti-Aβ42 toxic turn (24B3) (IBL, Gunma, Japan), 1:500 anti-ADAM10 (B-3) (IBL, Gunma, Japan), 1:140 anti-nELAV(HuD+HuC) (Santa Cruz; Santa Cruz, CA, USA), or 1:1000 anti-ECE1 (abcam; Cambridge, MA, USA). Following primary antibody incubation, blots were washed before being incubated with the appropriate secondary antibody. Blots were developed with enhanced chemiluminescence and quantified using Lumino Graph II (ATTO; Tokyo, Japan).