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. 2012 Jul 15;23(14):2635–2644. doi: 10.1091/mbc.E11-09-0805

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© 2012 Tao et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — Numbl interacts with TRAF5 in vivo and in vitro. (A) Purified GST-TRAF5 and GST proteins were incubated with His-Numbl in the presence of glutathione beads. The bound proteins were collected and analyzed by Western blotting with anti-His antibody. (B) HA-Numbl and myc-TRAF5 vectors (1.5 μg each) were cotransfected into HEK293T cells. Extracts with equal amount of protein were immunoprecipitated with anti-myc or anti-HA antibody and analyzed by Western blotting. (C) H4 cells were lysed with RIPA buffer and equal amounts of lysates were coimmunoprecipitated with anti-TRAF5 antibody. The normal rabbit immunoglobulin G was used as a negative control, and the TRAF6 antibody was used as a positive control. (D) U-87MG cells were fixed, and immunofluorescence microscopy was performed using the indicated antibodies to show colocalization of endogenous Numbl and TRAF. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole. Scale bar: 25μm. (E) Numbl contains a phosphotyrosine-binding domain (N1), a Phe-rich domain (N3), and a coiled-coil domain (N5). HEK293T cells were transfected with myc-TRAF5 and HA-Numbl mutants (1.5 μg each). Cell lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting. (F) TRAF5 contains a finger domain (T1), a zinc finger motif (T3), and a TRAF domain (T5). HEK293T cells were transfected with myc-TRAF5 mutants and HA-Numbl (1.5 μg each). Cell lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting.