Figure 2.

Coimmunoprecipitation of NMDA receptors with CaMKII. (A) To test whether NMDA receptors are associated with CaMKII in vivo, crude membrane fractions were prepared from total rat forebrain and NMDA receptors were solubilized with deoxycholate. After immunoprecipitation with antibodies against NR1, a mixture of the antibodies against CaMKIIα and β (19) or with control mouse IgG, immunoblotting was performed (lanes 1–3) with antibodies against NR2A (Left Top), NR2B (Left Middle), and NR1 (Left Bottom). To measure the effect of NMDA receptor-mediated Ca²⁺ influx on CaMKII association with NMDA receptors in intact neurons, acute cortical slices were prepared and treated under control conditions (vehicle) or with 200 μM NMDA for 5 min in the absence or presence of 50 μM MK801 or 50 μM KN62. Crude membrane fractions were prepared and solubilized with deoxycholate. Immunoprecipitations were performed with a mixture of the antibodies against CaMKIIα and β (19) before immunoblotting with αNR1 (Right Lower) or a mixture of antibodies against NR2A and NR2B (Right Upper) (lanes 4–7). Similar results were obtained in two other experiments; the results of all three cortical slice experiments were quantified by densitometry of the immunoblotting signals (33) and are summarized in B. Bars = SEM.