Pharmacological modulation of α-secretase activity alters AICD-Gal4 levels and AICD-Gal4 mediated luciferase activity in SY5Y-APP-Gal4 cells. (A) Stimulation of α-secretase by PMA (1 μM PMA for 10 hours) increases sAPPα, C83-Gal4, and AICD-Gal4 levels as detected by Western blot analysis. (B) Quantification of Western blot densitometry in panel A. Normalization for loading differences was achieved by dividing the densitometry values for individual bands by the densitometry values for β-actin in the same lane. (C) Dose-dependent increases of AICD-Gal4-mediated luciferase activity with increasing concentrations of PMA (10 hour incubation). Luciferase levels normalized to total cell number using protein concentration. (D) Inhibition of α-secretases by TAPI-1 (20 μM for two hours) results in decreases in sAPPα, C83-Gal4, and AICD-Gal4 levels as detected by Western blot analysis. (E) Quantification of Western blot densitometry in panel D and normalized β-actin levels in the same lane. (F) Dose-dependent decreases in AICD-Gal4-mediated luciferase activity with increasing TAPI-1concentrations (two hour incubation). For the luciferase experiments, points represent mean normalized luciferase activity (+/- standard error) of three independent trials, with luciferase levels normalized to total cell numbers using SYBR Green. Student's t-tests with sequential Bonferroni correction for multiple comparisons; * indicates p < 0.05; ** indicates p < 0.01. "Control" uses the same media as the treatments and contains the same amount of DMSO as drug treated cells.