Fig. 1.

Association of SR to brain membranes inhibits D-serine synthesis. (A) Western blot analysis showing the presence of SR in rat brain homogenate (H) and synaptosomal plasma membrane (SM). SR is not released from SM by washing once (1T) or twice (2T) with buffer containing 0.5% Triton X-100 (Upper). Middle and lower panels show the postsynaptic density protein 95 (PSD-95) and synaptophysin (Syn) levels. (B) SR is not removed from the membrane by high-salt washing. Fraction 1T was incubated in buffer containing 10 mM Tris-HCl (pH 7.4) and increasing concentrations of NaCl. Membrane-bound SR was recovered by centrifugation and analyzed by Western blot. (C) Membrane-bound SR is mostly inactive. Cytosolic (SR cyt) and membrane-bound (SR memb) SR were separated by centrifugation of cerebral cortex of adult rats (Left), primary neuronal cultures (Middle), and SH-SY5Y neuroblastoma cells overexpressing HA-SR (Right). The values of D-serine synthesis were normalized to the amounts of SR as determined by densitometric analysis of the Western blot with SR antibody. The results in A and B are representative of 3–5 experiments with different preparations. The values in C represent the average ± SEM of 5–6 experiments done in triplicates. ***Different from control at P < 0.001.