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. 2015 Mar 30;208(7):881–896. doi: 10.1083/jcb.201412085

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© 2015 Moudgil et al.

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Figure 4. — Inhibition of farnesylation does not affect CENP-E and CENP-F KT localization. (A and B) HeLa cells were treated with 10 µM of L744832 FTI or DMSO for 24 h, fixed, and immunostained for hSpindly, CENP-E or CENP-F, and ACA. CENP-E and CENP-F localized to KTs in FTI-treated HeLa cells, whereas hSpindly did not. (C and D) Normalized fluorescence signals for CENP-E and CENP-F at KTs with 24-h DMSO or FTI treatment. n = 20. Boxes represent interquartile distributions and whiskers represent 10th and 90th percentiles. FTI-treated cells have slight but not significant increase in CENP-E or CENP-F signal as compared with control DMSO as indicated by p-value. (E) A schematic diagram of hCENP-F depicting the minimal KT localization domain (2581–3210 aa) as previously shown by other studies and the C3207A mutation in the C-terminal farnesylation motif (+, KT localization; −, no KT localization). (F) HeLa cells were transiently transfected with EGFP-hCENP-F fusion constructs (shown in Fig. 3 E) and KT localization ability of each construct was analyzed using fluorescence microscopy. Top panel is EGFP-CENP-F^2581-3210 and bottom panel is C3207A mutant of the same fragment. The images show that the farnesylation motif of CENP-F is not required for KT localization. Bars, 10 µm.