Transfection and dual-luciferase reporter assay.

Neuro-2A cells (8 × 10⁵) were seeded into 60-mm dishes containing EMEM with 10% FCS at 24 h prior to transfection. Two hours before transfection, medium was replaced with fresh growth medium lacking any antibiotics. Cells were cotransfected with the designated plasmid containing the firefly luciferase gene downstream of the SV40 early promoter (0.25 μg of plasmid DNA) and a plasmid encoding Renilla luciferase under the control of a minimal herpesvirus thymidine kinase (TK) promoter (50 ng of DNA) as described in the legends to Fig. 3, ​,4C,4C, and ​and55 to ​to77 and Table 1. To maintain equal plasmid amounts in the transfection mixtures, the empty expression vector was added as needed. Neuro-2A cells were incubated in 2% charcoal-stripped fetal calf serum after transfection. At 24 h after transfection, Neuro-2A cultures were treated with water-soluble DEX (10 μM [D2915; Sigma]). Forty-eight hours after transfection, cells were harvested, and protein extracts were subjected to a dual-luciferase assay using a commercially available kit (E1910; Promega). Luminescence was measured by using a GloMax 20/20 luminometer (E5331; Promega).