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. 2009 Mar 9;184(5):647–657. doi: 10.1083/jcb.200811012

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© 2009 Lince-Faria et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 4. — Mad2 associates with and requires Mtor to localize to unattached KTs. (A and B) S2 cells treated with colchicine and processed for immunofluorescence with Mad2 (red) and CID (green) antibodies. DNA (blue) was counterstained with DAPI. (C) Quantification of Mad2/CID pixel intensity at KTs for control (median = 0.946, range = 0–5.52, n = 571 KTs/20 cells) and Mtor RNAi (median = 0.357, range = 0–3.95, n = 515 KTs/20 cells). The two populations are statistically different (P < 0.001; Mann-Whitney test). (D–E′) Mitotic progression in S2 cells stably expressing GFP–α-tubulin (green) and mRFP-Mad2 (red). Arrows indicate KTs. Red cytoplasmic aggregates likely correspond to misfolded mRFP-Mad2. (F and G) Control and Mtor RNAi cells were treated with colchicine and processed for immunofluorescence with Mps1 (red) and CID (green) antibodies. (H) Quantification of Mps1/CID pixel intensity at KTs for control (median = 2.06, range = 0.04–13.1, n = 384 KTs/20 cells) and Mtor RNAi (median = 0.91, range = 0–8.9, n = 391 KTs/20 cells). The two populations are statistically different (P < 0.001; Mann-Whitney test). (I) Co-IP of Mad2 with Mtor in lysates obtained from Drosophila embryos harvested between 0–3 h after egg laying. Time is given in minutes/seconds. Bars, 5 µm.