PP6 catalytic or regulatory subunit depletion stabilizes the Aurora A–TPX2 complex. (A and B) TPX2–Aurora A complexes were isolated from control and PPP6C si08 (A) or control, PPP6C si07, or SAPS1–3-depleted (B) cell lysates prepared in the presence or absence of phosphatase inhibitors (PP-Inh.). GFP antibodies were used as a negative control (Con.). Total lysates and immune-precipitated (IP) material were Western blotted or used for kinase assays as indicated. White lines indicate that intervening lanes have been spliced out. (C and D) TPX2–Aurora A complexes were isolated from control, PP2C A–, and PP2C B–depleted (C) or control and PP1C A–, B–, and C–depleted (D) cell lysates prepared in the presence or absence of phosphatase inhibitors. Total lysates and immune-precipitated material were Western blotted. (E) Purified Aurora A–TPX2 complexes were incubated with wild-type or catalytically inactive recombinant PP6 holoenzymes, PP6^WT and PP6^PD, respectively, buffer, mock-purified enzyme, or 2.5 U of calf intestinal phosphatase (CIP) for 2 h and then Western blotted. The graph shows phosphorylated Aurora A levels (n = 2). Error bars indicate the standard error of the mean. (F) A model outlining the role of PP6 as an Aurora A–TPX2 phosphatase. PP1 and PP2A can act as free Aurora A phosphatases but cannot recognize the TPX2-complexed form that promotes spindle formation. Where present, the red and black lines indicate the phosphorylated and nonphosphorylated forms of Aurora A. Molecular mass is given in kilodaltons.