Cortical RAB high-salt homogenates that had been retained prior to centrifugation as described above were utilized for immunoblot analyses of APP holoprotein, BACE1 and APP carboxy-terminal C99/C89 and C83 fragments. 0.1 ml of cortical homogenates were treated with 25 μl of 10% SDS to solubilize membrane proteins, followed by centrifugation at 100,000 × g for 20 min at 40C. The supernatant was collected, and the pellet was re-extracted with 0.1 ml of 10% SDS in water. After centrifugation, this supernatant was combined with the first supernatant sample, and the total protein determined by BCA. A total of 30 μg of protein from each study mouse underwent SDS-PAGE (4–12% gradient gels), followed by blotting to nitrocellulose membranes as previously described [50 (link)]. Intact APP holoprotein and carboxy-terminal APP fragments were detected using the carboxy-terminal APP monoclonal antibody 5685 [48 (link)], as previously described [50 (link)]. BACE1 was also detected (Abcam), and GAPDH (Advanced Immunochemical) was used as a loading control. Secondary antibodies and imaging were essentially as described [50 (link)], using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE). All APP, APP fragments and BACE1 integrated signals were normalized to GAPDH integrated signal for each sample. The quantification was conducted by an individual masked to the blot lane assignments.