Figure 10

Biochemical analysis of the interaction between Nlg and NMDA receptor-containing immuno-isolates. (A) NR2B and Nlg were immunoprecipitated from detergent solubilized P4 rat visual cortex and blotted with antibodies specific for NR1 and Nlg1. NR1 and Nlg1 were co-immunoprecipitated using these antibodies, but not by Protein-G sepharose beads alone (control). (B) Immunoprecipitations (IPs) from detergent-solubilized P4 rat visual cortex using antibodies to NR2B and Nlg did not co-immunoprecipitate the synaptic proteins synapsin1 and GluR2. (C) NR1-containing membrane fractions were immuno-isolated from P2–3 rat cortex. Western blots of these organelles reveal enrichment for many other postsynaptic proteins, including Nlgs 1, 2 and 3. Input and control lanes were included for comparison. Control lanes represent isolations performed without primary antibody to NR1 (beads were coated with control IgG). Comparison of NR1 and control lanes shows enrichment of the blotted protein in NMDA receptor transport packets (NRTPs). (D) Nlg1 remained associated with NR1 in the immuno-isolated vesicle fraction after solubilization with Triton X-100, while GluR2 is no longer recovered. (E) Nlg1 was not enriched after washing with a carbonate buffer at pH11, which removes associated, but not integral vesicle proteins. In comparison, GluR2 remains associated with NRTPs under high pH conditions.