Trichoplein binds and activates AurA. (A) AurA can bind trichoplein but not Odf2-β in HeLa cells. The cells were transfected with the indicated proteins. 24 h after the transfection, cell extracts were subjected to immunoprecipitation with anti-Flag. Before immunoprecipitation, a fraction of each cell extract was subjected to immunoblotting with anti-MBP (trichoplein) or anti-GST (Odf2-β) as an input. (B) AurA can be activated by trichoplein in HeLa cells. The cells were transfected with Flag-tagged AurA wild type or its kinase-dead mutant (KM) in addition to Myc-tagged trichoplein and/or HA-tagged Odf2-β. 24 h after transfection, cell lysates were subjected to immunoblotting with anti-pAurA, anti-Flag (AurA), anti-Myc (trichoplein), or anti-HA (Odf2-β). (C) AurA can directly bind trichoplein in vitro. GST pull-down assays were performed using the indicated purified proteins. GST or MBP was used as a negative control (−). (D) AurA can be directly activated by trichoplein in vitro. The kinase assays were performed as described in Materials and methods. Each reaction mixture was subjected to autoradiography (32P-H3 or 32P-AurA) or immunoblotting with anti-GST (AurA) or anti-MBP (MBP or MBP-trichoplein). CBB, Coomassie brilliant blue; IP, immunoprecipitation; Tricho., trichoplein; IB, immunoblot.