PBA Enhances APP Proteolysis Figure 10. PBA stimulates APP/Fe65 nuclear signaling. Na¨ıve N2a cells were transiently transfected with combinations of APP-HA and Fe65Gal4 (A and B) or with APP-Gal4 and Fe65 (C). Treatment with titrated PBA levels demonstrated that Fe65-Gal4 signaling significantly increases 2.5 fold with 5 mM PBA (p,0.005; student t-test of 0 and 5 mM PBA) (A). As maximal Fe65-Gal4 transcriptional signaling occurs at a specific APP:Fe65 ratio, N2a cells were transfected with titrated levels of APP. A 1:1 vector ratio of APP:Fe65 stimulated maximal signaling in all treatment conditions. PBA significantly increased the Gal4-reporter activity over that observed with the untreated cells (p,0.0001; two-way ANOVA). Thapsigargin, in contrast, attenuated Gal4-reporter activity in comparison to the untreated cells (p,0.001; two-way ANOVA) (B). The APP-Gal4 assay was employed to test the consistency of the PBA mediated stimulation of AICD/Fe65 nuclear signaling. 24 hours post-transfection with APP-Gal4 and Fe65 at a 1:1 vector ratio, the N2a cells were treated with 5 mM PBA for an additional 24 hours. Two vector concentrations were employed for these assays—low (50 ng) and high (500ng). At both vector concentrations, PBA elicited a significant 2-fold increase Gal4-luciferase activity (C) (p,0.005; student t-test). doi:10.1371/journal.pone.0009135.g010 PLoS ONE | www.plosone.org 12 February 2010 | Volume 5 | Issue 2 | e9135