To detect the localization of APP695-myc or C99-myc and sorLA, transfected cells were immunostained using monoclonal antibody 8E5 (raised against 444–592 residues of APP770; a gift from Elan Pharmaceuticals, San Francisco, CA) and rabbit N-terminal anti-SOL-sorLA antibody (1:2000) or N-terminal rab LA sorLA antibody (provided by Anders Nykjaer, University of Aarhus, Aarhus, Denmark) or monoclonal 13G8 antibody (raised against 676–695 residues of APP770; 1:500; gift from Elan Pharmaceuticals), monoclonal 9E10 anti-c-myc antibody (Abcam, Cambridge, UK) and rab C-terminal anti-sorLA (raised against 20 C-terminal residues of sorLA; provided by Claus Munck Petersen, University of Aarhus, Aarhus, Denmark). To detect the subcellular localization of BACE, cells transfected with BACE–V5 were immunostained with anti-V5 monoclonal antibody (Sigma, St. Louis, MO). Endogenous BACE was stained or immunporecipitated by rabbit anti-BACE N-terminal antibody (Calbiochem, La Jolla, CA).
Immunoprecipitation of sorLA was detected by rabbit anti-SOL-sorLA antibody, and C99 was detected by mouse 6E10 antibody (Signet, Dedham, MA). Western blots were performed using anti-V5 (BACE-V5)and rabbit SOL–sorLA antibody for detection.
Ab40 levels were determined by sandwich ELISA using BNT77 (anti-Ab 11–28) as the capture antibody and horseradish peroxidase-linked BA27 as detection antibodies (Takeda Chemical Company, Osaka, Japan).