Figure 2. NCAM accumulates in the presynaptic membrane. A, Recycling synaptic vesicles were labeled by incubating live NCAM+/+ neurons with antibodies against the lumenal domain of synaptotagmin 1 applied in the culture medium containing 47 mm K+. Neurons were then fixed with formaldehyde and colabeled with antibodies against NCAM, PSD95 and synaptophysin. Note the clusters of NCAM overlapping with accumulations of synaptotagmin 1 and synaptophysin apposed to PSD95 clusters (arrows). B, Formaldehyde-fixed NCAM−/− neurons transfected with NCAM140 were labeled with antibodies against NCAM, the presynaptic marker protein synaptophysin and the postsynaptic marker protein PSD95. An axon of an NCAM140 transfected neuron is shown. Note that accumulations of synaptophysin colocalize with accumulations of NCAM140 in NCAM−/− neuron transfected with NCAM140 abutting onto NCAM−/− neurons (arrows). NCAM140/synaptophysin accumulations are apposed to PSD95 clusters. C, Brain homogenates, synaptic plasma membranes (membranes) and synaptic vesicular organelles (vesicular organelles) were probed by Western blot with the antibodies against the intracellular domain of NCAM140 and NCAM180, synaptophysin (synapt.), PSD95, and Na,K-ATPase. Arrows indicate NCAM isoforms detectable by the antibodies. Note that NCAM accumulates in the synaptic plasma membrane. Synaptic membranes, containing both presynaptic and postsynaptic portions, are also enriched in PSD95 and plasma membrane localized Na,K-ATPase. NCAM is also present in synaptic vesicular vesicles, which are highly enriched in synaptophysin but are free of PSD95 and Na,K-ATPase. NCAM180 detectable in the synaptic vesicular organelles is probably contained in postsynaptic transport organelles that are present in this fraction. NCAM immunoreactivity at ∼160 kDa most likely is a proteolytic fragment of NCAM180. The graph shows quantitation of blots (mean + SEM; n = 3) with the signal in homogenates set to 100%. *p < 0.05, paired t test. D, Synaptosomes and highly purified synaptic vesicles (synaptic vesicles) were probed by Western blot with polyclonal antibodies against a common epitope at the C terminus of NCAM140 and NCAM180, monoclonal antibody D3 against an NCAM180-specific epitope in the intracellular domain of NCAM 180, and CSP. Arrows indicate NCAM isoforms detected by the antibodies. Note that NCAM140 is detectable in immunopurified synaptic vesicles. A smear above the NCAM140 band was reproducibly detectable in our preparations and may represent glycosylated NCAM140 or NCAM140-containing protein complexes, which were not fully dissociated by SDS-PAGE. Note that NCAM180 is not detectable in synaptic vesicles with NCAM180-specific antibodies even after prolonged exposure of the blot, while NCAM180 is readily detectable in synaptosomes. Mock immunopurification (mock) of synaptic vesicles with nonimmune Igs served as control of immunopurification. In these experiments, 4–12% polyacrylamide gels were used to enable detection of both NCAM isoforms and CSP in one SDS-PAGE run, which was necessary to control for protein loading and specificity of synaptic vesicle immunopurification, resulting in NCAM140 and NCAM180 labeling patterns different from the data shown in C, in which 8% polyacrylamide gels were used and low molecular weight proteins were allowed to run out of the gel to achieve better separation of the NCAM bands. Scale bars: 10 μm.