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Figure 6. HSET, Eg5, and cytoplasmic dynein are required for the formation of microtubule asters in a cell free mitotic extract. Specific antibodies were used to immunodeplete either Eg5 (ΔEg5; C and D), cytoplasmic dynein (ΔDynein; E and F), or both Eg5 and cytoplasmic dynein (ΔEg5/ΔDynein; G and H) from a HeLa cell mitotic extract. Untreated extracts (A and B) or the depleted extracts were then supplemented with preimmune antibodies (control) or HSET-specific antibodies (+HSET Ab). The formation of microtubule asters was stimulated by the addition of taxol, ATP, and by incubation at 31°C. The resulting structures were analyzed by indirect immunofluorescence using antibodies specific for tubulin and NuMA, as indicated. I, Eg5-, Dynein-, and Eg5/Dynein-depleted mitotic extracts supplemented with preimmune or HSET-specific antibodies were separated into 10,000 g soluble (S) and insoluble (P) components and the immune pellet fraction (PAb), and were subjected to immunoblot analyses using antibodies specific for Eg5, cytoplasmic dynein, and HSET, as indicated. Bar, 10 μm. Image published in: Mountain V et al. (1999) © 1999 The Rockefeller University Press. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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