chamber on the microscope stage. Imaging was performed using a 60× 1.4 oil immersion objective on a standard inverted microscope under the control of MetaMorph 7.5 software or a spinning-disk confocal system (Ultraview Vox; PerkinElmer). A maximum intensity projection of the fluorescent channels was performed. Images were then cropped in ImageJ (National Institutes of Health) and placed into Illustrator CS3 (Adobe Systems, Inc.) to produce the figures. Isolation of PP6 complexes Wild-type and catalytically inactive PP6 complexes were isolated from 3 × 15–cm dishes of si08ResPPP6CWT, si08ResPPP6CPD stable HeLa cell lines, or untransfected control cells grown to 60% confluence. The cells were ar- rested in mitosis with 100 ng/ml nocodazole for 16 h. Mitotic cells were harvested by shake off, washed three times with warm PBS, and then resus- pended in warm growth medium. The cells were incubated in a 5% CO2 incubator at 37°C for 20 min and then washed twice with cold PBS. The cells were lysed in 1 ml of cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% Triton X-100 with protease inhibitor cocktail), left on ice for 20 min, and then centrifuged at 20,000 gav for 20 min at 4°C to remove insoluble debris. To isolate PP6 complexes, 20 µl mouse anti-FLAG M2 agarose beads (A2220; Sigma-Aldrich) were added into 900 µl of cell ly- sate and rotated at 4°C for 3 h. The beads were washed with cell lysis buffer, and the PP6 complex was eluted with 2× 70 µl of 200 µg/ml FLAG peptide in TBS, pH 7.5, to give E1 and E2 fractions. Phosphatase and kinase assays Aurora A–TPX2 complexes for use as a substrate in in vitro phosphatase assays were prepared as follows from 3 x 15–cm dishes of HeLa cells grown to 60% confluence. Cell lysates were prepared as for PP6 isolation except that buffers contained phosphatase inhibitor cocktail 1 (Sigma- Aldrich). For Aurora A complex isolation, 2 µg mouse anti-TPX2 antibody and 60 µl protein G–Sepharose beads were added to 700 µl of cell lysate, and the mixture was rotated at 4°C for 2 h. The beads were washed with lysis buffer and then TBS and aliquoted into five tubes. To each tube, either 20 µl TBS, E1-eluted si08Res PPP6CWT or si08ResPPP6CPD complex, E1-eluted HeLa control, or TBS with 0.5 µl of calf intestinal phosphatase was added. The tubes were incubated at room temperature for 2 h and then boiled for 5 min at 95°C after adding 20 µl of 3× SDS loading buffer. Samples were analyzed by Western blotting on normal gels or gels containing 50 µM Phos-tag reagent (Wako Chemicals USA, Inc.) charged with Mn2+ to better resolve phosphorylated from nonphosphorylated forms of the same pro- tein. Quantitations were performed using ImageJ from blots of normal gels because reduced transfer efficiency of phosphorylated relative to non- phosphorylated forms of the same protein from Phos-tag gels was observed. For in vivo phosphatase assays, 5 × 10–cm dishes of HeLa cells were transfected with control or PPP6C si08 duplexes for 48 h and then treated with 100 ng/ml nocodazole for 16 h. Cells were harvested and lysed as for PP6 complex purification, except that the sample was split into two aliquots, one containing phosphatase inhibitor cocktail 1 and the other without. Cell lysis was left to proceed on ice for 20 min, and then insoluble debris was removed by centrifugation at 20,000 gav for 20 min at 4°C. A 30-µl aliquot of supernatant was kept on ice as an input sample, and Aurora A–TPX2 complexes were immune precipitated from the remaining lysate as described in the previous paragraph. The samples were analyzed by Western blotting, and measurements were performed using ImageJ. For phosphatase subunit siRNA library screening assays, this procedure was scaled down to use individual wells of a 6-well plate, and only cell lysates were analyzed. For in vitro kinase assays, a modification of a published method was used (Preisinger et al., 2005). Control or Aurora A–TPX2 complexes isolated from 1.6 × 106 cells were incubated in 50 mM Tris-HCl, pH 7.3, 50 mM KCl, 10 mM MgCl2, 20 mM -glycerophosphate, 15 mM EGTA, 100 µM ATP, 0.5 µl -[32P]ATP, and 1 µg histone H3 substrate in 20 µl of final volume for 30 min at 30°C. Reactions were analyzed by SDS-PAGE and autoradiography. Mass spectrometry Protein samples for mass spectrometry were separated on 4–12% gradi- ent NuPAGE gels and then stained using a colloidal Coomassie blue stain. Gel lanes were cut into 12 slices and then digested with trypsin (Wilm et al., 1996). The resulting tryptic peptide mixtures in 0.05% trifluoroacetic acid were then analyzed by online liquid chromatography with tandem mass spectrometry with a nanoAcquity UPLC system (Waters Corporation) and a mass spectrometer (Orbitrap XL ETD; Thermo Fisher Scientific) fitted with a nanoelectrospray source (Proxeon). Peptides were loaded onto a 5-cm × 180-µm trap column (BEH-C18 Symmetry; Waters Corporation) in 0.1% formic acid at 15 µl/min and then resolved using a 25-cm × 75-µm column using a 20-min linear gradient of 0 to 37.5% acetonitrile in 0.1% formic acid at a flow rate of 400 nl/min. The mass spectrometer was set to acquire a mass spectrometry survey scan in the Orbitrap (resolution = 30,000) and then perform tandem mass spectrometry on the top five mul- tiply charged ions in the linear quadrupole ion trap after fragmentation using collision ionization (30 ms at 35% energy). A 90-s rolling exclusion list with n = 3 was used to limit redundant analysis. Maxquant and Mascot (Matrix Science) were then used to compile and search the raw data against the human international protein index database. Protein group and peptide lists were sorted and analyzed in Excel (Microsoft) and Max- quant (Cox and Mann, 2008). Mass spectrometry and tandem mass spec- trometry spectra were manually inspected using Xcalibur Qualbrowser (Thermo Fisher Scientific). Online supplemental material Fig. S1 shows that there is redundancy between protein phosphatase catalytic subunits during mitosis. Fig. S2 shows that the subunits of the PP6 holoenzyme localize to the cytosol. Fig. S3 shows that kinetochore proteins localize normally and cold-stable kinetochore fibers are still pres- ent in PPP6C-depleted cells. Fig. S4 shows increased Aurora A pT288 at the spindle in PPP6C-depleted cells. Fig. S5 shows reduced levels of KIF11/Eg5 at the spindle in the absence of PPP6C. Videos 1 and 2 show the effect of PPP6C depletion on spindle formation and chromosome align- ment in living cells. Table S1 contains a list of the siRNA duplexes directed toward phosphatase catalytic and regulatory subunits used in the course of this study. Online supplemental material is available at http://www.jcb .org/cgi/content/full/jcb.201008106/DC1. We thank Anja Dunsch for commenting on the manuscript, Dean Hammond for help with MaxQuant, and Colin Hornby for initial assistance with live-cell confocal imaging. This work was supported by a Cancer Research UK career develop- ment fellowship to U. Gruneberg (C24085/A8296) and a Cancer Research UK program grant to F.A. Barr (C20079/A9473). Submitted: 18 August 2010 Accepted: 23 November 2010 References Adams, J.A. 2003. Activation loop phosphorylation and catalysis in pr-o tein kinases: is there functional evidence for the autoinhibitor model? Biochemistry. 42:601–607. doi:10.1021/bi020617o Afshar, K., M.E. Werner, Y.C. Tse, M. Glotzer, and P. Gönczy. 2010. Regulation of cortical contractility and spindle positioning by the protein pho-s phatase 6 PPH-6 in one-cell stageC. elegans embryos. Development. 137:237–247. doi:10.1242/dev.042754 Ahonen, L.J., M.J. Kallio, J.R. Daum, M. Bolton, I.A. Manke, M.B. Yaffe, P.T. Stukenberg, and G.J. Gorbsky. 2005. Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores. Curr. Biol. 15:1078–1089. doi:10.1016/j.cub.2005.05.026 Andrésson, T., and J.V. Ruderman. 1998. The kinase Eg2 is a component of the Xenopus oocyte progesterone-activated signaling pathway.EMBO J. 17:5627–5637. doi:10.1093/emboj/17.19.5627 Bamford, S., E. Dawson, S. Forbes, J. Clements, R. Pettett, A. Dogan, A. Flanagan, J. Teague, P.A. Futreal, M.R. Stratton, and R. Wooster. 2004. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br. J. Cancer. 91:355–358. Bayliss, R., T. Sardon, I. Vernos, and E. Conti. 2003. Structural basis of Aurora-A activation by TPX2 at the mitotic spindle.Mol. Cell. 12:851–862. doi:10 .1016/S1097-2765(03)00392-7 Bischoff, J.R., L. Anderson, Y. Zhu, K. Mossie, L. Ng, B. Souza, B. Schryver, P. Flanagan, F. Clairvoyant, C. Ginther, et al. 1998. A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers.EMBO J. 17:3052–3065. doi:10.1093/emboj/17.11 .3052 Bollen, M., D.W. Gerlich, and B. Lesage. 2009. Mitotic phosphatases: from entry guards to exit guides. Trends Cell Biol. 19:531–541. doi:10.1016/ j.tcb.2009.06.005 Chen, F., V. Archambault, A. Kar, P. Lio’, P.P. D’Avino, R. Sinka, K. Lilley, E.D. Laue, P. Deak, L. Capalbo, and D.M. Glover. 2007. Multiple protein phosphatases are required for mitosis in Drosophila. Curr. Biol. 17:293– 303. doi:10.1016/j.cub.2007.01.068 1330 JCB • VOLUME 191 • NUMBER 7 • 2010