Fig. 6.

Changes in NR2B subunit phosphorylation on Ser1480 during chronic ethanol exposure and withdrawal. Immunoprecipitation and Western blot analyses were used to determine the effects of chronic ethanol exposure and withdrawal on the phosphorylation state of Ser1480 on NR2B subunits. A and B, phosphoserine antibodies directed against NR2B Ser1480 detected a ∼170-kDa band in cultured hippocampal homogenates that had been precipitated with NR2B antibodies. This band was decreased in samples from chronic ethanol-treated cells (A) and returned to control levels shortly after ethanol was removed from the cultures (WD 15 min). Incubation with 10 μM TBB during ethanol withdrawal prevented the reversal to control phosphorylation levels (B). C, quantitative analysis of Western blots by densitometry revealed a significant decrease in Ser1480 phosphorylation after chronic ethanol exposure. Inhibition of CK2 with TBB resulted in a decrease in Ser1480 phosphorylation 15 min after withdrawal, relative to cells treated with ethanol alone. Phosphorylation levels were normalized to total NR2B band volume and are expressed as a percentage of untreated control. Asterisk indicates a significant difference from untreated controls, and @ indicates a significant difference between ethanol + TBB and treatment with ethanol alone (* or @, p < 0.01, pairwise comparison using one-way ANOVA adjusted by Holm-Sidak method; n > 4 blots for each condition).