Figure 2
ER stress induces APP degradation.
(A), 7w-PSML cells pretreated with MG132 or vehicle for 30 min were exposed to A23187 for 12 h. Cells were harvested for immunoblotting with anti-22C11 (APP) and anti-β-actin. The bottom panel shows a representative western blot and the top panel shows the quantification based on densitometry. Statistical significance was analyzed by one-way ANOVA followed by a Tukey's multiple-comparison test. (n = 4, **P < 0.01 versus control group; ##P < 0.01 versus A23187-treated group) (B, C), Pretreatment with MG132, a proteasome inhibitor, protects the APP protein from ER stress-mediated degradation. 7w-PSML cells were pre-treated with MG132 for 30 min followed by treatment with thapsigargin (B) or tunicamycin (C) for 12 h and harvested for western blot analysis to analyze expression levels of APP (22C11). (D), 7w-PSML cells were pretreated for 60 min with BAPTA/AM (5 μM) before a 12 h treatment with A23187 (1 μM). Chelation of the intracellular calcium with BAPTA prevented degradation of APP. Statistical significance was analyzed by one-way ANOVA followed by a Tukey's multiple-comparison test (n = 5, ***P < 0.001 and *P < 0.05). All the gels were run under the same experimental conditions. For each experiment, APP level was quantified by densitometry and normalized to β-actin loading control. Full-length images are presented in the supplementary information.
