Gas2 Inhibits Cell Division Figure 5. The Gas2 protein co-sediments with F-actin and microtubules. (A) The experimental flowchart of the cytoskeleton co- sedimentation assays. The samples were pre-treated with either 10 mM taxol, 20 mM nocodazole, 20 mM phalloidin, or 20 mM latrunculin B to study the cytoskeletal binding properties of Gas2. The samples were incubated on ice for one hour to de-polymerize microtubules. Taxol was added stepwise to a final concentration of 20 mM to polymerize tubulin into microtubules and samples were incubated at room temperature for one hour. Samples were loaded on the top layer of 30% sucrose solution in tubulin stabilization buffer (TSB), then centrifuged at 100,000 g for 30 minutes at room temperature. The pellets were re-suspended into the same volume as the supernate. Samples were run on SDS-PAGE for Western blot analysis. (B) The GFP-CH domain co-sediments with F-actin in the pellet. When F-actin was de-polymerized into actin monomers with latrunculin B, the CH domain was detected in the supernate (Fig. 5B: SL and PL). (C) The GFP-Gas2 domain co-sediments with microtubule in pellets. When microtubules PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24698