tralized by 1 M Tris-HCl, pH 9.5. The eluate was dialyzed against PBS CJ-023423 web containing 0.1% of N-octyl b-D-glucopyranoside. Immunoprecipitations and pull-down assay Cultured cells were washed three times with cold PBS, and then solubilized at 4uC for 30 min using RIPA buffer supplemented with complete protease inhibitor cocktail. Synaptosomes were incubated in the presence of 1% N-octyl b-D-glucopyranoside for 40 min at 4uC. For immunoprecipitation, the samples were centrifuged at 1,000 g and the resulting supernatants were incubated with antibodies for 3 h at 4uC. Fifty ml Protein A/G-suspension were added to the mixture and incubated overnight at 4uC. Beads were pelleted at 1,000 g and 4uC, washed three times with ice-cold PBS containing 1% N-octyl b-D-glucopyranoside, Triton-X100 or CHAPS for 10 min, and once with PBS. SDS sample buffer was added to the beads and the samples were boiled at 95uC for 5 min. The beads were pelleted by centrifugation and the supernatants were subjected to SDS-PAGE. Mass spectrometry Protein samples were subjected to SDS-PAGE followed by staining with the colloidal Coomassie blue staining Roti-Blue kit and stained protein bands were cut out 16365279 of the gel. After successive treatment with dithiothreitol and iodoacetamide, in-gel digestion of proteins by 5 ng trypsin/ ml in 50 mM NH4HCO3 was carried out overnight at 37uC. Gel pieces were then repeatedly extracted with 50% acetonitrile/5% formic acid, and the combined extracts were dried down in a vacuum concentrator, re-dissolved in 5% methanol/5% formic acid, desalted on a Calreticulin Regulates APP Processing Purified GST or the GST fusion proteins were bound to glutathione-agarose beads and incubated with cell lysates or the respective membrane fractions overnight at 4uC on a rocking platform. Beads were pelleted at 1,000 g, washed three times with lysis buffer and once with PBS at 4uC. Bound proteins were eluted by incubation with 40 mM glutathione. b Amyloid ELISA kit and 
Human/Rat b Amyloid ELISA kit as described in the manufacturer’s protocol. Immuno-isolation of 10712926 the c-secretase complex For isolation of intact c-secretase complexes, HEK cells stably transfected with mutated APP and wild type or mutated presenilin-1 were used. Cells were homogenized at 4uC in MOPS buffer propanesulfonic acid, pH 7.0, 10 mM KCl) containing protease inhibitor cocktail. After centrifugation for 10 min at 1,000 g and 4uC, the supernatant was further centrifuged for 45 min at 100,000 g. The pelleted membranes were incubated with CHAPSO lysis buffer dimethylammonio]-2-hydroxy-1-propanesulfonate, pH 6.4, 150 mM sodium citrate) containing protease inhibitor cocktail for 30 min on ice and centrifuged for 30 min at 100,000 g and 4uC. After preclearing, the supernatant was incubated with protein A/G agarose and the monoclonal presenilin-1 antibody`Nixon for 2 h at 4uC. Immunoprecipitates were obtained by centrifugation at 2,000 g for 5 min and 4uC, washed 4 times with CHAPSO-wash buffer containing protease inhibitor cocktail, and resuspended in 0.2% CHAPSO, 35 mM sodium citrate, pH 6.4, 3.5% glycerol, 30 mM DTT, 0.5 mg/ml L-aphosphatidylcholine, protease inhibitor cocktail. Binding assays For ELISA, proteins or peptides were immobilized on a polyvinylchloride surface in 96-well-plates at concentrations of 510 mg/ml overnight at 4uC. The following steps were carried out at room temperature. After washing five times with PBS, wells were blocked by adding 2% BSA in PBS for 1 h. After washing