Decrease in APP-CTFα levels in CHO cell lines stably expressing prodomain mutant forms of ADAM10. CHO-APP-ADAM10 stable cell lines were treated with 250 nM DAPT for 24 h to inhibit γ-secretase and APP-CTFs (C83, C89, C99) levels were examined in total cell lysates. (A) Representative western blots. (B) Graphs for relative CTFα levels normalized with full-length APP levels (C83/FL-APP) and further with ADAM10 levels (C83/FL-APP/ADAM10). n = 7, mean ± SEM. Significance of changes in the mutant cells was calculated when compared with wild-type. *P < 0.05, **P < 0.005 (two-tailed Student's t-test). EV, empty vector-transfected cells; W, wild-type ADAM10 cell; M11, M12, Q170H mutant cells; M21–M23, R181G mutant cells; DN1, DN2, E384A dominant negative mutant cells.

Attenuated PMA-inducible α-secretase activity in CHO cells stably expressing the prodomain mutant forms of ADAM10. Each CHO-APP-ADAM10 stable cell line was treated with 1 µM PMA or equivalent amount of DMSO for 6 h and harvested to monitor sAPPα levels. (A) Representative western blots. (B) Graphs for relative sAPPα levels. n = 4–6, mean ± SEM. **P < 0.005 (two-tailed Student's t-test).

Decrease in constitutive α-secretase activity in CHO cells stably expressing APP and wild-type or prodomain mutant forms of ADAM10. CHO-APP₇₅₁ cells were used to generate cell lines stably overexpressing different ADAM10 constructs, wild-type (W), Q170H mutant (M11, M12), R181G mutant (M21–M23), E384A dominant negative mutant (DN1, DN2) and empty vector (EV). Each stable cell line was grown to exponential phase and conditioned media and cell lysates were collected to measure levels of sAPPα, ADAM10 and APP. (A) Representative western blots. FL-APP, full length APP; imm-ADAM10, immature ADAM10; mat-ADAM10, mature ADAM10. (B) Graphs for relative sAPPα levels normalized with full-length APP levels (sAPPα/FL-APP) and further with ADAM10 levels (sAPPα/FL-APP/ADAM10). n = 4–8, mean ± SEM. (C) Restoration of the reduced sAPPα levels in ADAM10 mutant cells by overexpression of wild-type ADAM10. Each ADAM10 stable cell line was transiently transfected with wild-type ADAM10 construct and levels of sAPPα and ADAM10 were measured. (D) Ratios of mature and immature ADAM10. Mature and immature ADAM10 bands in the western blot in (A) were quantified and their ratio in each cell was calculated. n = 3–6, mean ± SEM. Significance of changes in the mutant cells was calculated when compared with wild-type. *P < 0.05, **P < 0.005 (two-tailed Student's t-test).

Increase of Aβ₄₀ levels in the prodomain mutant ADAM10 stable cell lines. CHO-APP-ADAM10 stable cell lines were grown to exponential phase. Twenty four hour after changing media, the conditioned media were collected and levels of Aβ_x-40 were measured by ELISA. Aβ₄₀ levels were normalized with amount of total protein in each corresponding cell lysate. n = 6–12, mean ± SEM. Significance of differences of the mutant and empty-vector transfected cells versus wild-type was calculated. *P < 0.05, ***P < 0.0005 (two-tailed Student's t-test). EV, empty vector-transfected cells; W, wild-type ADAM10 cell; M11, M12, Q170H mutant cells; M21–M23, R181G mutant cells.