Hippocampal neurons were pretreated with 50 or 75 mM ethanol for 15 min; 100 μM carbachol was then added to some of the samples (solid bars) and cells were incubated for an additional 30 min. Cell lysates were collected, proteins were quantified, separated, transferred to PVDF membranes and incubated with phospho-ERK1/2, ERK1/2, or β-actin antibodies as described in Materials and Methods. (A) Average optical density of phosphorylated ERK1/2 normalized to controls (mean ± SEM; n=3; *p<0.05 compared to control; #p<0.05, compared to carbachol alone). (B) Representative immunoblot derived from a membrane labeled with phosphoERK1/2 (pERK), total ERK1/2 (tERK), and β-actin (loading control) antibodies.