and following by centrifugation. Only the cytosolic layers were used for co-sedimentation assays. The samples were separately pre-treated with 10 mM taxol (Sigma), 20 mM nocodazole (Calbiochem); and 20 mM phalloidin (Calbiochem), and 20 mM latrunculin B (Calbiochem) to investigate Gas2 cytoskeleton binding properties. The mixture samples were incubated on ice for 1 hour to de-polymerize microtubules, and then taxol was step wisely added to the final concentration 20 mM to polymerize tubulin into microtubules filaments at room temperature for one hour. Samples were loaded on the top layer of 30% sucrose in tubulin stabilization buffer (TSB: 1 mM EGTA, 5 mM MgCl2, 80 mM K-PIPES at pH 7.0), and then they were centrifuged at 100,000 g for 30 minutes at room temperature. The pellets were diluted into equal volume as the supernatant samples. Samples were run on 10% SDS-PAGE for Western blot analysis. The following antibodies were used for the Western blot analysis: the monoclonal mouse tubulin antibodies (Sigma, DM1A) were used at 1:3000 dilution; the polyclonal rabbit actin antibodies (Biomedical Technologies Inc. BT-560) were used at 1:3000 dilution; the monoclonal mouse GFP antibodies (Roche, Clone 7.1 and 13.1) were used at 1:3000 dilution. Primary antibodies were detected using IgG antibodies conjugated to HRP (Molecular Probes) at 1:3000 dilution by ECL method. Electron microscopy Purified actin (AKL99-A) and tubulin (TL238-A) were bought from Cytoskeleton Inc. (Denver, CO.). Actin was first dissolved in the general actin buffer (5 mM Tris-HCl at pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP), and then phalloidin (Calbiochem) was added up to final concentration 20 mM to polymerize actin into F- actin. Tubulin was first dissolved into general tubulin buffer (80 mM PIPES at pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP), and then 20 mM final concentration taxol (Sigma) was used to polymerize tubulin into microtubules. The purified full-length Gas2 protein in PBS was mixed with F-actin or microtubules or both for EM examination. A similar experiment was done by References 1. Grinnell F, Petroll WM (2010) Cell motility and mechanics in three-dimensional collagen matrices. Annual review of cell and developmental biology 26: 335–361. 2. Mandato CA, Bement WM (2003) Actomyosin transports microtubules and microtubules control actomyosin recruitment during Xenopus oocyte wound healing. Current biology: CB 13: 1096–1105. 3. Heng YW, Koh CG (2010) Actin cytoskeleton dynamics and the cell division cycle. The international journal of biochemistry & cell biology 42: 1622–1633. 4. Rodriguez OC, Schaefer AW, Mandato CA, Forscher P, Bement WM, et al. 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Bement WM, Mandato CA, Kirsch MN (1999) Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes. Current biology: CB 9: 579–587. 11. Zhang T, Mandato CA (2007) Xenopus oocyte wound healing as a model system for analysis of microtubule-actin interactions. Methods in Molecular Medicine Microtubule Protocols: Humana Press. pp 181–188. Gas2 Inhibits Cell Division adding the full-length Gas2 to actin or tubulin samples before their polymerization. For each EM sample, 5 ml protein sample was placed on a glow discharged pre-carbon coated copper grid for 1 minute, and then was replaced by 5 ml 1% uranyl acetate for 1 minute treatment. Uranyl acetate was carefully removed and the treated grid was left by air-dried. Samples were examined in an FEI Tecnai 12 electron microscope, and digital images were taken by a Gatan 792 Bioscan wide angle multiscan CCD camera. Supporting Information Video S1 BSA-injected Xenopus embryos cell division time-lapse movie. The movie was recorded at 1 frame per minute and plays at 10 frames per second. (MOV) Video S2 Gas2-injected Xenopus embryos cell division time-lapse movie. The movie was recorded at 1 frame per minute and plays at 10 frames per second. (MOV) Acknowledgments We would like to thank Prof. Jacque Paiement at University of Montreal for providing the experimental Gas2 antibody; Prof. Marko Horb at Institut de recherches cliniques de Montreal for providing the experimental frogs; Dr. Wolfgang E. Reintsch for helping on the cyro technique; Cell Imaging and Analysis Network at McGill University for using the confocal microscope, and Facility for Electron Microscopy Research at McGill University for using the electron microscope. Author Contributions Conceived and designed the experiments: TZ CAM. Performed the experiments: TZ BD IR CAM. Analyzed the data: TZ. Wrote the paper: TZ IR EJL CAM. 12. 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(2009) p53-dependent translational control of senescence and transformation via 4E- BPs. Cancer cell 16: 439–446. PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e24698