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. 2015 May 15;26(10):1845–1856. doi: 10.1091/mbc.E14-11-1560

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© 2015 Holland et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Tvwo months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Farnesylation of Spindly is required for its localization to microtubule-unattached kinetochores. (A–C) HeLa cells immunostained for the kinetochore proteins Spindly (A), CENP-E (B), and CENP-F (C) after treatment for 48 h with 10 μM farnesyltransferase inhibitor FTI-277 or DMSO. Cells were incubated in 1 μM nocodazole for 4 h to maximize the accumulation of the proteins at kinetochores and costained with anti-centromere antibodies (ACAs). Scale bars, 5 μm. (D) Quantification of protein levels at kinetochores in the conditions shown in A–C using immunofluorescence intensity measurements. Each condition represents a total of 100 kinetochore measurements from 20 different cells. Error bars represent the SEM with a 95% confidence interval. The t test was used to determine statistical significance (***p < 0.0001). (E, F) HeLa cells immunostained for the kinetochore proteins Zwilch (E) and Hec1 (F) after treatment for 48 h with 10 μM farnesyltransferase inhibitor FTI-277 or DMSO. Scale bars, 5 μm. (G) Kinetochore level quantification of the conditions in E and F displayed as described for D (ns, not statistically significant). (H) Kinetochore localization of RNAi-resistant, MycGFP-tagged wild-type (WT) and mutant (C602S) Spindly in nocodazole-treated HeLa cells after depletion of endogenous Spindly, visualized by immunofluorescence with an anti-GFP antibody (see Figure 5A for corresponding RNAi immunoblot). Scale bar, 5 μm. (I) Kinetochore level quantification of the condition in H displayed as described for D.