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Figure S1. Analysis of nectin-1 point mutations from amino acid residues 301 to 351. HEK 293 cells were transfected with each mutant. Twenty-four hrs after transfection, cells were lysed in reducing sample buffer and analyzed on 12% SDSPAGE. Samples were transferred to nitrocellulose, and the blots were probed with antinectin-1 cyto-specific antibody. All experiments were repeated five times.

Figure S2. Analysis of nectin-1 point mutants in neuro-2A cell lines. Neuro-2A cells were transfected with nectin-1 point mutations that alter nectin-1 processing in HEK 293 cells. The cells were collected in reducing sample buffer 24 hrs after transfection and analyzed by Western blotting. The blot was probed with antinectin-1 cyto-specific antibody.

Figure S3. Nectin-1 localizes to both pre- and postsynaptic sites. A. Neurons at 28 DIV were examined at the ultrastructural level by electron microscopy. Synapses were identified by the presence of thickened presynaptic and postsynaptic specializations with intervening dense material indicated by arrows. Nectin-1 immunostaining was observed both in pre- and postsynaptic sites using antibody against the intracellular domain of nectin-1. Over 30 synapses were examined and the immunoreactivity of nectin-1 was observed in all synapses. B. Nectin-1 immunostaining was abrogated by incubation of the antibody with its cognate antigenic peptide.

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