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. 2006 Feb 13;172(4):505–515. doi: 10.1083/jcb.200510028

Figure 5.

Figure 5.

The carboxy-terminal domain of NEDD1 binds to γTuRCs, and the amino-terminal domain mediates centrosome attachment. (A) Transient overexpression of GFP fusion proteins with full-length NEDD1 (GFP-NEDD1) and amino acids 1–319 (GFP-NEDD1Nt) and 321–660 (GFP-NEDD1Ct) in U2OS cells. Cells are stained for γ-tubulin (red) and DNA (blue). Arrows indicate the positions of centrosomes that are shown enlarged in insets, displaying the fluorescence of the various GFP fusion proteins or γ-tubulin. For comparison, an untransfected cell is indicated by an arrowhead in the left panel (inset in top right corner shows an enlarged view). Bar, 10 μm. (B) Mitotic U2OS cells overexpressing GFP-NEDD1Nt or GFP-NEDD1Ct (green) that were stained for α-tubulin (red) and DNA (blue). GFP-NEDD1Ct induces spindle defects. Bar, 5 μm. (C) Immunoprecipitation of the overexpressed proteins by anti-GFP antibody in Cos-7 cells. (top) Detection of precipitated proteins by anti-GFP antibody (note that GFP is resolved in a 10% polyacrylamide gel, whereas the other proteins are separated in a 7.5% gel). (bottom) γTuRC proteins (γ-tubulin, GCP2, and GCP4) coprecipitate with GFP-NEDD1 and GFP-NEDD1Ct. (D) Schematic drawing depicting functional domains of NEDD1.