TPX2 phosphorylation by Aurora A is required for intact bipolar spindle assembly in mitotic Xenopus egg extract. (A) Mitotic extracts (CSF) depleted of pEg2 (Aurora A) or xTPX2 were subjected to Western blotting. Histone H3 Serine10 phosphorylation serves as a mitotic marker. (B) Coomassie blue staining showing the recombinant proteins added in C. (C and D) CSF depleted of xTPX2 or pEg2 was added with indicated proteins. Sperm-induced bipolar spindle formation was impaired by xTPX2 or pEg2 depletion. Among all proteins to rescue such defects, GFP-xTPX2-3D showed the best effect, whereas GFP and GFP-xTPX2-3A showed no rescue. GFP-xTPX2 could partially rescue xTPX2 but not pEg2 depletion; His-pEg2 could rescue pEg2 but not xTPX2 depletion. Bar, 20 µm. Three independent experiments were performed in D, each measuring 100 spindles. Error bars indicate SD.