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Figure S3. The centromere-targeting domains of CENP-B and CENP-C are necessary but not sufficient for stable binding to centromeres in living cells. (A) Colocalization experiment of the DNA-binding domain (DBD; amino acids 2–169; Shelby, R.D., K.M. Hahn, and K.F. Sullivan. 1996. J. Cell Biol. 135:545–557) of CENP-B fused to EGFP with endogenous CENP-A demonstrates proper centromere localization of this construct. (B) Short-term FRAP experiment of EGFP–CENP-B–DBD in HEp-2 cells. Circles indicate areas of bleaching and recovery. (C) Quantitation of the fluorescence recovery over time for GFP–CENP-B–DBD in interphase and mitotic cells in comparison to FRAP of the full-length EGFP–CENP-B protein. (D) Colocalization experiment of an EGFP-tagged deletion variant of CENP-C containing its central DNA-binding domain (amino acids 315–635; Lanini, L., and F. McKeon. 1995. Mol. Biol. Cell. 6:1049–1059; Yang, C.H., J. Tomkiel, H. Saitoh, D. Johnson, and W.C. Earnshaw. 1996. Mol. Cell. Biol. 16:3576–3586; Sugimoto, K., K. Kuriyama, A. Shibata, and M. Himeno. 1997. Chromosome Res. 5:132–141). Note that this construct, besides binding to centromeres, also decorates nucleoli diffusely. (E) Short-term FRAP experiment of EGFP–CENP-C(315–635) in HEp-2 cells. Circles indicate areas of bleaching and recovery. (F) Quantitation of the fluorescence recovery over time of EGFP–CENP-C(315–635) compared with full-length EGFP–CENP-C. (G) Colocalization experiment of an EGFP-tagged deletion variant of CENP-C containing the C-terminal centromere-targeting sequence (amino acids 635–943; Trazzi, S., R. Bernardoni, D. Diolaiti, V. Politi, W.C. Earnshaw, G. Perini, and G.D. Valle. 2002. J. Struct. Biol. 140:39–48) with CENP-A. (H) Short-term FRAP experiment of EGFP–CENP-C(635–943) in interphase HEp-2 cells. Circles indicate areas of bleaching and recovery. (I) Quantitation of FRAP of EGFP–CENP-C(635–943) compared with full-length EGFP–CENP-C. Recovery curves represent mean values from at least 10 measurements. The standard deviation in FRAP experiments was <10% in all cases. Bars, 5 µm.