Fig. 2.

Chronic ethanol exposure increases the C2′/C2 ratio of NMDAR NR1 splice variants. Immunoprecipitation and Western blot analyses were used to determine the effects of chronic ethanol exposure and withdrawal on the expression of C2′ and C2 splice variants of NMDAR-NR1 subunits in cultured hippocampal neurons. A, splice variant-selective antibodies directed against NR1-C2′ or -C2 detected ∼120-kDa bands in cultured hippocampal homogenates after immunoprecipitation with monoclonal NR1 antibodies. Band intensity of C2′-containing subunits increased, whereas C2-containing subunits decreased after 7-day exposure to ∼80 mM ethanol. In whole homogenates, bands detected with antibodies against neuronal tubulin did not change with ethanol exposure. B, quantitative analysis of Western blots by densitometry revealed a significant increase in the ratio of C2′ to C2 detected after chronic ethanol exposure. Band intensities were normalized to neuronal tubulin band volume as a loading control. Asterisk indicates a significant difference from untreated controls (*, p < 0.01, comparison using Student's t test; n > 3 blots for each condition).