To immu noprecipitate Ab, 7WD10 cells were grown in six well plates and were treated with BCNU at final concentra tions of 0, 0. 5, 1. 0, 5. 0, 10. 0 and 20. 0 uM in duplicate wells. After 48 hours, the CM was collected, centrifuged to remove cell debris and immunoprecipitated overnight using a monoclonal Ab9 antibody to pull down sellectchem total Ab. After SDS PAGE electrophoresis using NuPAGE 4% to 12% bis tris gels, total Ab was detected by immunoblotting using a mixture of 6E1082E1 antibodies which reliably detects total Ab as described previously. The CM was also immunoblotted to detect sAPPa, sAPPb and sAPPtotal using the indicated antibodies. To detect APP holoprotein and CTFs, the cells were lysed using lysis buffer with complete protease inhibitor mix and equal amounts of proteins were loaded into each well and subjected to SDS PAGE electrophoresis.

Following transfer onto poly vinylidene difluoride membranes, they were blocked with 5% milk in Tris buffered saline with Tween and incubated overnight with primary antibo dies followed by one to four hours of incubation with horseradish peroxidase conjugated secondary anti bodies, such as monoclonal mouse Inhibitors,Modulators,Libraries anti goat IgG light chain or monoclonal mouse anti rabbit IgG light chain. The protein signals were detected using Super Signal West Inhibitors,Modulators,Libraries Pico Chemiluminescent substrate. Quanti tation of Western blot signals was done using Java based ImageJ software available freely from NIH. APP turn over and surface biotinylation Inhibitors,Modulators,Libraries experiments Near confluent 7WD10 cells in duplicate wells were trea ted with BCNU at 10.

0 mM concentration and after 24 hours, washed two times with cold PBS and incubated with 2. 0 mgml sulfo NHS LC biotin in PBS, pH 8. 0 under ultra low shaking on ice in the cold room. After an hour of incubation, cells were washed Inhibitors,Modulators,Libraries three times in PBS and lysates were prepared using 1% Nonidet P 40 lysis buffer containing complete protease mix as described above. Biotinylated proteins were pulled down by immu noprecipitation with anti biotin antibody plus anti mouse agarose beads. The samples were subjected to SDS PAGE and APP was detected with CT15 antibody. To assess the effect of BCNU on APP stability, cyclohexi mide experiments were done essentially as previously described in our published papers. Briefly, 7WD10 cells were incubated with cycloheximide at a concentration of 100 mgml in PBS at 37 C in a CO2 incubator.

Treated and untreated cells were harvested and lysed Inhibitors,Modulators,Libraries at 0, 15, 30, 60, and 120 minutes. The lysates were processed to detect APP with CT15 antibody as described above. Measurement of activities of secretase enzymes Enzyme activities were measured using partially purified enzymes from mouse brain homogenates furthermore using commer cially available kits for BACE and g secretase according to the manufacturers instructions.