CTFs were detected by Perifosine Western blotting using CT15 anti body. Similar to Ab levels, CTF levels were also signifi cantly decreased at 5. 0 uM, 10. 0 uM and 20. 0 uM compared to untreated controls. Thus, except for the first two lower concentrations, BCNU significantly decreased APP processing and CTF production, starting from 5. 0 uM. These changes occurred without alterations in APP holoprotein which was also detected by the CT15 antibody. The decreased CTF levels are consistent with decreased Ab levels by BCNU and indicate that BCNU decreases amyloidogenic processing of APP in CHO cells. BCNU significantly increases levels of sAPPa and sAPPtotal but not sAPPb To obtain an overall picture of APP metabolism under BCNU treatment, we further quantified the levels of secreted large extracellular N terminal domain truncated protein at the a site or at the b site as well as levels of sAPPtotal from the same CM used for Ab quantification.
More than a one fold increase was noted Inhibitors,Modulators,Libraries for sAPPa levels at some concentrations of BCNU applied for 48 hours, although we did not notice a strict dose dependent increase in sAPPa levels. The secretion Inhibitors,Modulators,Libraries of sAPPa was increased to 167% at 0. 5 uM, 186% at 1. 0 uM, 204% at 5. 0 uM and 152% at 10. 0 uM. For some unknown reason, sAPPa levels were not altered at the highest concentration of 20. 0 uM tested, although Ab levels were significantly decreased. The maximum increase was noted at 5. 0 uM concentration. Similarly, the levels of sAPPtotal were increased by 159% at 5. 0 uM, 172% at 10. 0 uM and 170% at 20. 0 uM in BCNU Inhibitors,Modulators,Libraries treated cells.
On the other hand, there were no significant alterations in the levels of sAPPb at any of the BCNU concentrations tested. The sAPPa levels were detected using 6E10 antibody which recognizes an epitope within the 1 17 amino acid of the Ab domain of APP. sAPPtotal was detected Inhibitors,Modulators,Libraries by the 63G antibody whose epitope lies within the mid region of APP. Taken together, decreased levels of Ab and CTFs and increased release of sAPPa and sAPPtotal in the CM clearly indicate that BCNU may decrease amyloidogenic processing of APP by increasing a secretase mediated cleavage of APP thereby reducing the APP substrate avail able for cleavage by BACE enzyme. BCNU increases immature APP at the cell surface As endocytosis of surface APP is required for Ab genera tion, we next examined whether BCNU treatment altered surface levels of APP.
Inhibitors,Modulators,Libraries To do this, all surface pro teins were labeled with biotin and immunoprecipitated with antibiotin antibody. Results showed that BCNU treated cells accumulated immature APP by more than one selleck chemicals llc fold at the cell surface compared to untreated cells, while there was no change in the surface levels of mature APP. Next, to test whether decreased amyloidogenic processing of APP is due to changes in turn over of APP, we performed cyclo heximide chase experiments.