NEDD1 residues 572–660 interacts with γ-tubulin directly.

<p>(A) The interaction of endogenous NEDD1 and γ-tubulin was assessed in HEK293T cells. Due to its low level of expression, NEDD1 is not detectable in the inputs (1/20 lysates loaded), however γ-tubulin is present (first lane). Both NEDD1 and γ-tubulin are detected in lysates immunoprecipitated with NEDD1 antibody (second lane). NEDD1 is not detected when γ-tubulin is immunoprecipitated (third lane). Additional bands are IgG. In negative controls, γ-tubulin and NEDD1 are not detected when an unrelated antibody (HSP70) (fourth lane) or no antibody (fifth lane) was used for the immunoprecipitation. (B) Full length NEDD1 (660 aa), or two truncation constructs (1–571 and 572–660) were fused to a Myc-tag at their N-terminus. (C) The interaction of full length and truncated forms of Myc-NEDD1 with endogenous γ-tubulin was assessed in HEK293Ts. Expression is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with full length (660 aa) NEDD1, and 572–660 NEDD1, but not 1–571, using a Myc antibody. * represent the correct size for each construct. When no antibody is added (negative controls), no γ-tubulin is immunoprecipitated. (D) The interaction of NEDD1 CTD (residues 572–660) and γ-tubulin was assessed <i>in vitro</i>. Recombinant GST or GST-NEDD1 CTD bound to glutathione sepharose beads were incubated with His-γ-tubulin, with or without HEK293T lysate. Inputs (1/5 lysates loaded) are shown in lanes 6–8. Endogenous γ-tubulin is expressed in the lysate (lane 9). After incubation with the beads and removal of unbound proteins, γ-tubulin is not bound to GST alone (lane 1), but is bound to GST-NEDD1 CTD both in the absence and presence of lysate (lanes 2 and 5 respectively). Endogenous γ-tubulin in the lysate does not to bind to GST alone (lane 3), but does bind to GST-NEDD1 CTD (lane 4).