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Fig. 1. Immunisation against the NTD of the NMDA receptor NR1 subunit prevents both the interaction of tPA with NR1 and subsequent cleavage of NR1 in vivo. (A) Schematic representation of the hypothesis: antibodies raised against the NTD-NR1 should interact with endogenous NR1 and thus prevent the potentiating effect of tPA on NMDA receptor signalling. (B) 1, 5 or 10 µg of recombinant N-terminal domain of NR1 (NTD-NR1, amino acids 19-371) was separated by SDS-PAGE prior to detection with sera collected from NTD-NR1- or CFA-immunised mice. Immunoblot is representative of three independent experiments performed with sera from three different animals for each group. (C) tPA activity in cortical and striatal extracts measured by zymography assay in CFA-treated mice and vaccinated mice (n=3). (D) Evidence of IgG immunoreactivities in the cerebral parenchyma of NTD-NR1- or CFA-immunised mice (representative photomicrographs taken from the same anatomical level of the cortex) (E) Protein extracts from cerebral cortices of control and immunised mice were subjected to immunoprecipitation against tPA or NR1 before immunoblotting against NR1 as described in the Materials and Methods section. Immunoblot is representative of three independent experiments. IB, immunoblot; IP, immunoprecipitation; CFA, CFA-injected; NTD, NTD-NR1-injected.
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