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. 2004 Mar 1;18(5):541–558. doi: 10.1101/gad.1151204

Figure 8.

Figure 8.

Syntenin binds Unc51.1 and Rab5. (A) The C′-tail domain of Unc51.1 (amino acids 829–1051) and that deleting the last three amino acids (VYA–; amino acids 829–1048) were tested for their ability to bind the full-length Syntenin as well as its deletion mutants (N that spans the N′-terminal half of Syntenin, and 2PDZs that spans the two PDZ domains of Syntenin) in a β-galactosidase filter assay. Unc51.1 C′-tail displayed very strong binding (+++) toward the full-length Syntenin and the two PDZ domains of Syntenin. (B) COS7 cells were cotransfected with the HA-tagged full-length Syntenin and the myc-tagged full-length Unc51.1 or the Unc51.1 with VYA deletion. Immunoprecipitation was done with an anti-HA antibody, and the immune complex was analyzed with SDS-PAGE followed by immunoblot with an anti-myc antibody. The HA blot indicates the presence of Syntenin in the immune complex. (C) COS7 cells were cotransfected with the myc-Unc51.1 and HA-Syntenin constructs, fixed, and double-stained with a monoclonal myc (green) and a polyclonal HA antibodies (red). Bar, 10 μm. (D) The full-length Rab5 was tested for its ability to bind the full-length Syntenin as well as deletion mutants (N and 2PDZs) in a β-galactosidase filter assay. Rab5 displayed very strong binding (+++) toward the full-length Syntenin and the two PDZ domains of Syntenin. (E) COS7 cells were transfected with the myc-tagged full-length Unc51.1 and the myc-tagged Rab5 with or without the HA-tagged Syntenin. Immunoprecipitation was done with an anti-Rab5 antibody, and the immune complex was analyzed with SDS-PAGE followed by immunoblot with an anti-myc and an anti-HA antibodies. COS7 cells were cotransfected with the HA-tagged full-length Syntenin and the myc-tagged full-length Unc51.1 or the Unc51.1 with VYA deletion. Immunoprecipitation was done with an anti-HA antibody, and immune complex was analyzed with SDS-PAGE followed by immunoblot with an anti-myc antibody. The HA blot indicates the presence of Syntenin in the immune complex in a transfection-dependent manner. (F) Membrane-enriched fractions extracted from P6 cerebellum were immunoprecipitated with either the anti-Rab5 antibody or the normal rabbit IgG, and the resulting immune complexes were analyzed with SDS-PAGE followed by immunoblot with an anti-Rab5 antibody and an anti-Unc51.1 antibody.