Effects of PSD-95 on calpain-mediated cleavage of the NR2A subunit. HEK293 cells were transfected with NR1a, NR2A, and PSD-95, calpastatin, or vector control and treated with 100 μm glutamate and 100 μm glycine in the presence or absence of 100 μm MK-801 for 0 or 30 min. NMDA receptor activation resulted in a significant decrease in full-length NR2A immunoreactivity (A, representative Western blots of NR2A) as confirmed by quantification of NR2A levels (B) (N-terminal antibody A-6475; ^*p = 0.0008; n = 8). Coexpression of PSD-95 or calpastatin inhibited the reduction in NR2A immunoreactivity. No proteolysis of NR2A was observed when NMDA receptor activity was blocked by addition of MK-801. Quantitated data are expressed as a percentage of the MK-801 control condition. C, Calpain activity was measured after 30 min agonist treatment by assessment of a calpain-generated spectrin degradation product as detected by an antibody to this product (AB38); coexpression of PSD-95 had no effect on the production of calpain-cleaved spectrin compared with the vector control (p = 0.2441; n = 5). D, No difference was observed between intracellular calcium responses of NR1a/2A cells cotransfected with PSD-95 or vector control (p = 0.1709; n = 14). Data are shown as mean ± SEM (error bars).