Centrosome localization ability and the tubulin-binding domain of centrobin map to its C terminus (aa 765–903). (A–D) Centrobin interacts with tubulin in vitro, and the tubulin-binding domain maps to aa 365–903. GST or GST-centrobin and its mutants were incubated with HeLa cell tubulin at 4°C. The bound proteins were fractionated and immunoblotted with anti–α-tubulin antibody. Input, an aliquot of 10% tubulin used in the binding reactions. The input of purified GST or GST-centrobin and its mutant proteins (C and D) were stained by Coomassie blue. Molecular mass is indicated in kilodaltons. (E) A schematic representation of centrobin and its mutants summarizing their binding with α-tubulin. (F) Centrobin 765–903 localizes to centrosomes but not the microtubules. HeLa cells transfected with GFP-centrobin 765–903 were stained with anti–centrin 3 and anti–α-tubulin antibodies to visualize the centrioles (panel a) and microtubules (panel b), respectively. (G) GFP–centrobin 765–903 or centrobin-TuBD does not inhibit the microtubule nucleation process. HeLa cells transfected with control or centrobin-TuBD vectors were subjected to aster formation assay and stained with anti–α-tubulin. Quantification of different sizes of asters is presented. Histograms are plotted as mean ± SEM (n = 3). Bars, 1 µm.