For the NGFI-A plasmid, we subcloned the EGR1 coding sequence into a TOPO–His expression vector (pEF6/V5-His TOPO TA Expression kit, Invitrogen) 4. In co-transfection studies, human embryonic kidney HEK293 cells were plated at a density of 6 × 104 in six-well dishes and transiently co-transfected with a total amount of 1.5 µg of plasmid DNA (1.0 µg of NR3C1 promoter ligated into the pGEM-luc plasmid and 0.5 µg of NGFI-A expression plasmid or 0.5 µg of control pEF6/V5 plasmid) using the calcium phosphate method. HEK293 cells were maintained as a monolayer in DMEM (Invitrogen) containing 10% fetal calf serum (Colorado Serum Company). The cells were harvested 72 h after transfection and lysed, and luciferase activity was assayed using the Luciferase Assay System (Promega) according to the manufacturer’s protocol.
