site. This final step resulted in the tricistronic Gal4-lucifera- seEGFP/EF1-NLS-bGal vector that was used to generate the stable N2a reporter cells (Figure 1). Generation of APPGV16/Gal4-LuciferaseEGFP/EF1-NLS- bGal Stable c-Secretase Reporter Cells The EF1-APPGV16 expression vector described previously [5] contains a neomycin resistance element. The Gal4-lucifera- seEGFP/EF1-NLS-bGal construct contains a zeomycin resistance element. 10 mg of EF1-APPGV16 and Gal4-luciferaseEGFP/EF1- NLS-bGal were transiently co-transfected into a confluent 10 cm plate of na¨ıve neuroblastoma N2a cells. Two days post- transfection the N2a cells were passaged and placed under dual selection with 500 mg/ml G418 and 200 mg/ml Zeocin (Invitro- Figure 1. c-secretase dependence of the APPGV16/Gal4- luciferaseEGFP/EF1- NLS-bGal reporter cells. The N2a EF1- APPGV16/Gal4-luciferaseEGFP/EF1-NLS-bGal stable cells (NAG cells) have c-secretase dependent EGFP expression, as DAPT eliminates the majority of EGFP fluorescence (A). The Gal4-luciferase moiety of the reporter is also c-secretase dependent as titrated DAPT diminishes luciferase activity (B). The decrease in luciferase activity correlates with elevated CTFGV16 protein levels across the DAPT titration (C). doi:10.1371/journal.pone.0009135.g001 PBA Enhances APP Proteolysis gen). These cells were maintained under selection for approxi- mately three weeks, at which point individual clones were isolated and cultured in single wells of a 24 well plate. Once the clones were confluent, they where split, passaged, and screened for DAPT-sensitive luciferase activity and constitutive bGal activity by standard luminometric assays. The clone used in this study was selected due to high levels of c-secretase dependent (DAPT sensitive) luciferase activity and consistent bGal activity. The cells were expanded and continuously grown under selection. The N2a APPGV16/Gal4-luciferaseEGFP/EF1-NLS-bGal (NAG) cells were employed throughout the work presented here. Cell Culture Conditions The NAG cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) (Hyclone) at 37C in 10% CO2. The stock plates were maintained with 500 mg/ml G418 and 200 mg/ml Zeocin to maintain stability of the genetic elements. The experiments were performed when the NAG cells were between 50 and 75% confluent. Comparisons between the Gal4-transacti- vation levels and protein levels were done within the same experiment, unless otherwise indicated, wherein experimental conditions were staged in quadruplicate. Three wells were used per conditions for Gal4-transactivation assays, and 1 well was utilized for western blotting. In the experiments employing immunohistochemical approaches, the NAG cells were plated on 18mm coverslips coated with poly-D-lysine in 12 well plates. The transactivation assays and western blots were performed 24 hours following treatment unless otherwise indicated. Western Blots and Transactivation Assays The NAG cells were passaged from 60–80% confluent stock plates, and arrayed on 24 well plates for transactivation assays and western blotting. The transactivation assays were performed by lysing the NAG cells in 0.1% TX-100 lysis solution and were frozen at 280C to ensure complete lysis. The Gal4-luciferase and bGal luminometric assays were performed as previously described [5,29] employing a Berthold EG&G MicroLumat LB 96V luminometer. The output from the luminometric assays were point to point normalized, dividing the raw luminometric output from the luciferase assay by the bGal value for each sample. This data is plotted as Gal4-luciferase/bGal in the figures to denote that the raw luciferase data is normalized to constitutive reporter expression. The transactivation assays were performed in triplicate, and the standard deviation was calculated based upon the normalized value for each sample in each condition. The lysates for western blotting were generated using standard 1XRIPA buffer supplemented with 1X Protease Inhibitor Cocktail (Sigma). The lysates were rotated for 10 minutes at 4C, and spun for 30 minutes at 10,000g at 4C. The supernatant was mixed with 5X loading buffer and the blots were run using the NuPage Novex 4–12% gradient Bis-Tris gels (Invitrogen) with MOPS running buffer (Invitrogen). The blots were transferred to 0.2 mM nitrocellulose and all primary antibody incubations were per- formed at 4C overnight. Immunohistochemistry NAG cells were arrayed on poly-D-lysine coated coverslips in 12 well plates. The cells were treated as indicated and probed with the designed antibodies. The images were collected on either a Zeiss Axioskop 2 fluorescent microscope (Figure S1), or on a Leica SP1 laser scanning confocal microscope. The images were taken adjusting all settings to the untreated condition. All the settings were held constant for the remainder of the images to maintain accurate relative fluorescence between conditions. The primary PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9135