Prior to experiments, neurons were cultured for 14 days in 96-well plates (20,000 cells/well), 6-well plates (1 million cells/well), or T75 cm2 flask (5 million cells/flask). For all activity assays, neuronal cell pellets collected from EtOH/Ach treatment (17.5 mM EtOH or 10 μM Ach) with/without specific inhibitor for 48 h were sonicated in 50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 150 mM NaCl, and 20% glycerol after scraping off adherent cells without proteolytic enzymes. Cytosolic proteins (10-40 μg/replicate) collected from sonicated samples at 10,000 g centrifugation were used for activity assays.
