Cells were stimulated once with alcohol (at the indicated concentrations) at the start of each time course experiment or once each day (with fresh media) for the 4 day studies. Alcohol concentrations in media were determined with the alcohol testing kit (Pointe Scientific, Canton, MI). For example in the long term (4 day) studies, we found that after addition of alcohol each day alcohol concentration declined from 0.2% (43mM) over 24h to reach undetectable (zero) concentration. Thus, in order to expose the cells to alcohol during the 4 day experiments, we change media each morning and added fresh media with alcohol every day during the 4 day experiments. This paradigm models cellular alcohol exposure through alcohol in the blood circulation during daily and regular drinking by alcoholics. Nuclear extracts were prepared using a nuclear extraction kit (Pierce Biotechnology, Rockford, IL). Signaling experiments were terminated by removal of media and addition of PBS for scrapping, SDS/RIPA buffer for whole cell lysates, or nuclear extract kit buffer.
