Western blotting is shown of total proteins isolated from brain tissue following cycloheximide treatment of mice for the indicated times. (A) Transgene-derived human and endogenous APP in Tg2576 mice and endogenous murine APP in wild-type mice was detected with the anti-C-terminal APP antibody C1/6.1; β-tubulin, which is known to be stable in the CNS [52], is shown in control Western blots. An untreated wild-type mouse (wt) is included in the Tg2576 analysis to show the increased APP expression in this model. (B) Quantification of APP turnover in the Tg2576 and wild-type mice normalized to the band density without cycloheximide treatment; two-way ANOVA was used to compare APP turnover in the Tg2576 and wild-type mice (p<0.001). (C) Following the indicated cycloheximide treatment times, sAPP total was detected with 22C11 in Tg2576 mice. Human sAPPα and sAPPβ were detected by Western blotting with 6E10 and 6A1, respectively, and endogenous murine sAPPα and sAPPβ were detected by Western blotting with m3.2 and 242, respectively. Non-specific bands detected by the affinity purified polyclonal 242 are shown by the APP knockout brain homogenate. (D) Levels of endogenous sAPP total, sAPPα and sAPPβ were detected by Western blotting in wild-type mice following cycloheximide treatment as described above. The graphic representations in (E) compare the quantification of the levels of sAPP total in Tg2576 and wild-type mice (p<0.001). In (F), the turnover of the indicated sAPP species in the Tg2576 mice is shown (p<0.001, comparing murine sAPPα to the other sAPP species). In (G), the levels of endogenous sAPPα and sAPPβ in wild-type mice is shown (p>0.5). Throughout, quantifications are from two experiments (mean ± SEM) as specified in the Methods section.

(A) Western blots are shown using an antibody that recognizes tau independent of its phosphorylation status (T57120, top panels) and an antibody that detects a phospho-epitope on tau (PHF-1, bottom panels) in Tg2576 mouse brain and in wild-type mouse brain. Total brain proteins were analyzed as in Figure 1. (B) Change in the intensity of the T57120 signal following cycloheximide treatment in Tg2576 and wild-type mice is shown (mean ± SEM). (C) Change in the intensity of the PHF1 signal following cycloheximide treatment in Tg2576 and wild-type mice is shown (mean±SEM). Signal intensity is normalized to the PHF1 signal in wild-type mice without treatment (inserted Western blot). (D) Tau levels detected with T57120 and PHF-1 antibodies in wild-type mouse brain following cycloheximide treatment when mice were housed at ambient temperature (22°C) or at 34°C for 19 hours. β-tubulin is shown as a loading control. (E) Tau levels in primary neurons were detected by Western blotting with T57120 and PHF-1 antibodies after cycloheximide treatment as indicated. β-tubulin is shown as a loading control.

Neprilysin and IDE turnover is shown by Western blotting in Tg2576 (A) and wild-type mice (B); β-tubulin is shown as a control. (C) The ratio of DEA-extractable to formic acid-extractable human Aβ40 and Aβ42 in Tg2576 mice were measured by sandwich ELISA at the indicated times following cycloheximide treatment (mean ± SEM). (D) Turnover of endogenous DEA-extractable Aβ40 and Aβ42 levels in wild-type mice (mean ± SEM).