Fig. 2. TPX2 can bind to the importin /ß complex, an interaction that prevents TPX2 binding to Aurora A, but not microtubules. (A) The amino terminal domain of TPX2, TNT or BSA was coupled to Affiprep 10 beads, incubated with mitotic HeLa cell extract in the presence or absence of the importin /ß complex and GST-RanL43E, then re-isolated and analyzed by SDS PAGE and immunoblotting to detect importin ß. (B) GST-TNT was incubated with Taxol-stabilized microtubules in the presence or absence of the recombinant GST-RanL43E and or the importin /ß complex. Microtubules were re-isolated by centrifugation and the subsequent pellet (P) and supernatant (S) fractions analyzed by SDS-PAGE and immuoblotting to detect GSTTNT. (C) S-tagged TNT was incubated with mitotic HeLa cell extract in the presence of absence of GST-RanL43E and the importin /ß complex. TNT was recovered from the extract with S-protein agarose beads and analyzed by SDS PAGE and immunoblotting to detect Aurora A.