Fig. 1. Taxol-stabilized microtubules bind to NuMA from Xenopus egg extracts, irrespective of dynactin or dynein inhibition. (A) Anti-NuMA immunoblot of egg extract, and of supernatants (sup.) and pellets from extracts containing taxol-stabilized microtubules. Extracts were either untreated (left), or treated with recombinant dynamitin (middle), or monoclonal antibody against dynein intermediate chain (right). The cell cycle does not affect binding of NuMA to microtubules. (B) Anti-NuMA immunoblot; comparison of taxol microtubule pellets and supernatants from metaphase-arrested egg extract with equivalent material from extract released into interphase by calcium chloride. The taxol-treated extracts were supplemented with additional microtubules to increase the pelleting efficiency. Identical percentages of supernatants and pellets were loaded. NuMA in phosphatase-treated extract still binds to microtubules. (C) Anti-NuMA immunoblot of egg extract (extract), supernatant (taxol S.) and pellet (taxol P.) after centrifugation of taxol-treated extract, and the same pellet treated with either control buffer (b.contr.) or lambda phosphatase (-pptase). The phosphatase treatment consistently led to an increased immunoreactivity of NuMA on western blots (identical amounts were loaded for phosphatase treatment and controls, as verified by Ponceau staining in multiple experiments). The phosphatase-treated pellet was subsequently resuspended and re-centrifuged. The resulting supernatant (S.2) and pellet (P.2) are shown.