CHC associates with phospho-S558 TACC3. (A) The SYPRO ruby gel shows CHC pulled down from Noc-treated HeLa cell extracts by recombinant GST-TACC3 522–577 fusion proteins phosphorylated by recombinant aurora A. CHC peptides detected by mass spectrometry are indicated. (B) Western blotting shows CHC pulled down by recombinant GST-TACC3 proteins phosphorylated by aurora A. Input represents the 5% amount of Noc-treated HeLa cell extracts subjected to the pull-down assays. Coomassie blue staining shows various GST fusion proteins used for each binding reaction. The phosphorylation levels of GST-TACC3 proteins in kinase reactions are shown by autoradiography. (C–E) Western blots show complex formation of endogenous TACC3 and CHC by immunoprecipitation (IP) with the indicated antibodies from mitotic HeLa cells synchronized by Noc (C), from Noc-synchronized cells with or without additional treatment of 2 µM VX-680 (D), or from Noc-synchronized cell lysates with or without treatment of λ-phosphatase (λPPase) before being subjected to immunoprecipitation (E). The phospho-T288 level correlates to aurora A kinase activity. Input represents the 5% amount of the indicated lysates for each immunoprecipitation. Black lines indicate that intervening lanes have been spliced out. (F and G) Representative images of HeLa cells in metaphase stained with DNA (blue) and antibodies against α-tubulin or CHC (green) and TACC3 (H-300) or phospho-TACC3 (pTACC3; red) are shown. Bars, 10 µm.