The NFκB-dependent reporter plasmid p(κB)5-Luc, a gift from Dr. N. Mackman (Scripps Research Institute, La Jolla, CA) contains five tandem copies of the NFκB site from the human TNF-α gene. RAW264.7 macrophages either exposed to alcohol for 4 days or control macrophages were transfected with the reporter plasmids 5×NFκB-firefly luciferase and Renilla-Luciferase as control (Promega) using the transfection agent FuGENE 6 (Roche Applied Science) as reported earlier (20). After 24 h of incubation, control cells were treated with 25 mM alcohol in the presence or absence of LPS (100 ng/ml) or chronic alcohol exposed macrophages were exposed to LPS for 8 h and luciferase activity assessed with Dual Glo Luciferase Assay reagent (Promega) according to the manufacturer’s instructions. NFκB transcriptional activity as detected by firefly luciferase activity was normalized with the Renilla luciferase activity. The relative light units represent an average of triplicate samples.
