Neonatal DRG neurons and PC12 cells were plated onto coated 96-well plates (Nunc, Naperville, IL) as previously described [
14]. In either case, each sample was composed of 50 μL of Opti-MEM
® I (Cell Culture Facility, UCSF) combined with 0.70 ng of Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) and allowed to incubate for 5 minutes at room temperature. 50 μL of Opti-MEM
® I was also combined with a total of 0.3 μg/well of the desired pGL3-reporter construct and/or appropriate expression construct. Additionally, the reference
renilla luciferase reporter plasmid, pRL-SV40 was included at 0.05 μg/well. The Lipofectamine™ 2000 and DNA solutions were then combined following the manufacturer's recommendations (Invitrogen, Carlsbad, CA). Overall, transfection efficiency was < 5% in primary neonatal DRG neurons [
14]. When indicated, PC12 cells were cultured in the presence of NGF (100 ng/ml) after transfection. Following 48 hours of culture, cell lysates were prepared according to the manufacturer's recommendations of the Dual Luciferase Reporter Assay System
® (Promega, Madison, WI). Both firefly and renilla luciferase products were measured in a MicroLumatPlus
® LB96V microplate luminometer using Winglow
® software (Perkin-Elmer Berthold, Wellesley, MA). Firefly luciferase activity was normalized to renilla luciferase activity as a relative ratio resulting in a "Relative Luciferase Activity", which represents the transcriptional activity directed by a particular luciferase reporter construct. In experiments where multiple expression plasmids were required, empty control plasmid was used to maintain an equivalent DNA concentration between transfected samples.