Figure 3. Expression of NR2A and NR2B in the spinal cord and forebrain of PSD-93 knock-out mice. A, Immunoblots of PSD-93, NR2A, and NR2B proteins in total extracts of spinal cord and forebrain of wild-type (+/+), heterozygous (+/-), and knock-out (-/-) mice. PSD-93 protein is undetectable in knock-out mice. There was no significant difference in the density of NR2A or NR2B bands among wild-type, heterozygous, and knock-out mice (p > 0.05; n = 5 for each group). Tubulin was used as a loading control (data not shown). B, The expression and distribution of NR2A/2B immunoreactivity in the spinal cord of wild-type (+/+) and PSD-93 knock-out (-/-) mice. The density and localization of NR2A/2B immunoreactivity in the spinal cord of wild-type mice are similar to those in knock-out mice. Scale bar, 50 μm. C-E, Effect of PSD-93 deletion on surface NR2A and NR2B expression in the cultured neurons of spinal dorsal horn. The amount of sample loaded for the total (T) was 10% of that for the surface (S). C, The top panel depicts a representative Western blot that shows samples of total and biotinylated surface NR2A in wild-type (WT) and PSD-93 knock-out (KO) mice. The bottom panel shows the statistical summary of the densitometric analysis. Average surface NR2A levels in knock-out mice were reduced by 64% of the value in wild-type mice (*p < 0.05). D, The top panel depicts a representative Western blot that shows samples of total and biotinylated surface NR2B in wild-type (WT) and PSD-93 knock-out (KO) mice. The bottom panel shows the statistical summary of the densitometric analysis. Average surface NR2B levels in knock-out mice were reduced by 50% of the value in wild-type mice (*p < 0.05). E, The top panel depicts a representative Western blot that shows samples of total and biotinylated surface GluR1 in wild-type (WT) and PSD-93 knock-out (KO) mice. The bottom panel shows the statistical summary of the densitometric analysis. There was no significant difference in surface GluR1 expression between wild-type and knock-out mice (p > 0.05). F, G, Effect of PSD-93 deletion on surface NR2A and NR2B expression in the spinal cord neurons from adult mice. Surface expression was assayed by using a membrane-impermeable cross-linking agent (BS3). The cross-linked product is unable to enter polyacrylamide gels, so only the intracellular pool is resolved by Western blotting. Changes in surface expression are detected by observing changes in this indirectly detected intracellular pool. F, A representative Western blot that shows samples of total and intracellular NR2A, NR2B, and α-actinin, respectively, in the untreated control and BS3-treated groups of wild-type (WT) and PSD-93 knock-out (KO) mice. G, The statistical summary of the densitometric analysis. Average percentages of intracellular NR2A and NR2B levels in knock-out (KO) mice were increased by 190 and 194%, respectively, of the value in wild-type (WT) mice (*p < 0.05). In contrast, the average percentage of intracellular α-actinin level in knock-out mice was decreased by 0.92% of the value in wild-type mice.