Figure 6.

Polyclonal antibodies against NSPA. (A) Immunoblots (IB) with human α-hP₁₁ autoantibodies and rabbit anti-NSPA (α-NSPA) antibodies of carbonate-extracted synaptosomal membranes. Both antibodies recognize the high molecular mass protein (p331/NSPA) in total the synaptosomal membranes (Syn; lanes 1 and 4) and pellet (P; lanes 2 and 5) but not the supernatant (SN; lanes 3 and 6) of carbonate extraction. (B) Immune cross reaction of NSPA with α-hP₁₁ and α-NSPA antibodies. The p331/NSPA protein immunoprecipitated (IP) by α-hP₁₁ from extracts of a synaptosomes (lane 1) is recognized in immunoblot by α-NSPA but not by the preimmune serum (PI; lane 2). (C) Double immunofluorescence with α-NSPA and α-hP₁₁ antibodies. Nonpermeabilized N2a cells and cortical neurons in primary culture both show complete cell-surface colocalization of α-NSPA (red) and α-hP₁₁ (green) staining. All of these results indicate that α-NSPA and α-hP₁₁ antibodies recognize the same protein. Bar, 10 μm.