Primary human neurons cultured in 6-well plates for 14 days were preincubated with actinomycin D (100 ng/ml) or cyclohexamide (10 μg/ml) for 15 min prior to EtOH/Ach (17.5 mM EtOH or 10 μM Ach) treatment for 24 h. Cell pellets were used for protein or RNA extraction. For Western blot analyses, cell lysate proteins were extracted with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5) containing 20% glycerol, 5 mM MgCl2, 0.5 mM DTT, and protease inhibitor cocktail, while total RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA) for NOX and XOX mRNA level analyses. Isolated RNA was reverse-transcribed with random hexamers (Promega, Madison, WI). Real-time quantitative PCR was performed with cDNA using an ABI PRISM 7000 sequence detector (Applied Biosystems, Foster City, CA). NOX2, XOX (gene symbol XDH), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed using genespecific primers and TaqMan probes that are available as TaqMan gene expression assays Hs00166163_m1, Hs00166010_m1 (for NOX2, XOX), and Hs02758991_g1 (GAPDH), respectively (Applied Biosystems). Gene expression was normalized to human GAPDH used as an endogenous control.
