Figure 2. In NMDAR complexes of synaptosomes, C-terminally truncated NR2A subunits assemble with NR1 and NR2B. A, Subcellular fractions of synaptosome preparations (Hom., homogenates; Cyto., cytosolic; Micro., microsomal; Syn., synaptosomal) from forebrains of wild-type (left) and NR2AΔC/ΔC (right) mice were analyzed in immunoblots for the presence of NR1, NR2B, NR2A, PSD-95, calnexin, and β-actin. β-Actin was used to control that equal amounts of protein (10 μg) were loaded on the gel. PSD-95, NR1, NR2B, and NR2A are enriched in synaptosomes, and calnexin is enriched in microsomes. Synaptosomes of NR2AΔC/ΔC mice showed impaired enrichment for NR2AΔC compared with NR2A of wild-type mice. B, NMDAR complexes of synaptosome fractions were immunoprecipitated (IP) with antibodies recognizing the C terminus of NR1 and NR2B or the N terminus of the NR2A subunit. Ten percent of the input and immunoprecipitated samples were analyzed in immunoblots for the presence of NR1, NR2B, and NR2A subunits. Immunoblots that were performed with the same antibody used in the IPs are boxed and marked by a star. These IPs show a threefold to fivefold stronger signal intensity than the 10% input, indicating an IP efficacy of 30-50% depending on the amount of antibody used in the reaction. Immunoblots for the coassembled subunits of immunoprecipitated NMDAR complexes show that 5-10% of the precipitated subunit remain associated with coassembled subunit during the immunoprecipitation (for NR2A IPs of NR2AΔC/ΔC mice; see Results). In synaptosomes of both wild-type and NR2AΔC/ΔC mice, the anti-NR1 IP copurified the subunits NR2A and NR2B, the anti-NR2B IP copurified subunits NR1 and NR2A, and the anti-NR2A IP copurified subunits NR1 and NR2B. The normalized ratio of the copurified NMDAR subunits was used to evaluate their relative levels within NMDAR complexes of wild-type and NR2AΔC/ΔC mice.