Residues 599–660 of NEDD1 interact with γ-tubulin and prevent it from localizing to the centrosome.

<p>(A) GFP-tagged NEDD1 expression constructs used in the study. (B) Interaction of NEDD1 and γ-tubulin was assessed with endogenous γ-tubulin and GFP-tagged NEDD1 expression constructs in HEK293T cells. The expression of γ-tubulin and all NEDD1 constructs is confirmed in the inputs (Inputs, 1/20 lysates loaded). All GFP-NEDD1 constructs can be immunoprecipitated with a GFP antibody (IP: GFP). However, γ-tubulin (arrow) is only immunoprecipitated with full length (1–660), 572–660 (CTD), 586–660 and 599–660 NEDD1. The upper bands in the γ-tubulin immunoblot are IgG. (C) NIH-3T3 cells were transfected with various GFP-NEDD1 constructs and immunostained for GFP (green) and γ-tubulin (red). Each image contains representative transfected and non-transfected cells. The box on the right is an enlargement of the γ-tubulin stained centrosomes in the transfected cells. Full length GFP-NEDD1 (1–660 aa) localizes to the centrosome and does not alter γ-tubulin levels at the centrosome. No other GFP-NEDD1 fusion proteins are detected at the centrosome. GFP-NEDD1 CTD and 599–660 prevent γ-tubulin from localizing to the centrosome, whereas GFP-NEDD1 (1–571) has no affect on γ-tubulin at the centrosome. All other constructs which are not able to bind to γ-tubulin have no effect (611–660 shown as an example). Scale bars = 10 µm.