Xenbase Image ID: 118869

Figure 3. TACC3 is required for mitosis-specific microtubule assembly around centrosomes. (A) Microtubules were visualized by the addition of fluorescently labeled tubulin in control mock-depleted (left) and TACC3-depleted extracts (right). Centrosomes were added to interphase extracts (top), and then the extracts were driven into M phase by the addition of nondegradable cyclin B (bottom). Bar, 10 μm. (B) Quantification of tubulin fluorescence in A. (C) Immunoblots of depleted extracts. The extracts were immunodepleted with control IgG (lanes 1 and 2; mock dep) or anti-TACC3 antibody (lanes 3–6; Δ TACC3) with adding back of TACC3 (lanes 5 and 6) or control buffer (lanes 1–4). The blots of interphase (lanes 1, 3, and 5) and mitotic extracts (lanes 2, 4, and 6) were probed by anti-TACC3 (top; TACC3) and anti-XMAP215 (bottom; XMAP215). (D) Microtubules assembled around mitotic centrosomes in immunodepleted extracts with add back of recombinant proteins. Purified recombinant TACC3 or control buffer were added in mock-depleted or TACC3-depleted extracts. Bar, 10 μm. Insets show immunolocalization of TACC3 on centrosomes. Bar, 1 μm. (E) Quantification of tubulin fluorescence in D. (F) Inhibition of MCAK in TACC3-depleted extracts. Microtubules were assembled around centrosomes in mock-depleted extracts plus control buffer (left), TACC3-depleted extracts plus control buffer (middle), or anti-MCAK antibody (right). The addition of inhibitory MCAK antibody rescued the effect of TACC3 depletion on microtubule assembly around mitotic centrosomes. Bar, 10 μm. (G) Quantification of tubulin fluorescence in F. (B, E, and G) Relative fluorescence intensity of labeled tubulin around mitotic centrosomes (within 21.079 μm radius) was measured. Values shown are the means plus SD. Image published in: Kinoshita K et al. (2005) Copyright © 2005, 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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