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. 2016 Nov 4;7:13465. doi: 10.1038/ncomms13465

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Copyright © 2016, The Author(s)

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(a) Immunofluorescence with anti-CENP-A antibody in RbAp48 ON and OFF cells. Bar, 10 μm. (b) Quantification of levels of CENP-A, CENP-C and CENP-T at kinetochores in RbAp48 ON and OFF cells based on Immunofluorescence analysis. Error bars represent s.d.. Asterisk indicates statistically significance (P<0.0001) by Student's t-test. (N=100). (c) Top: outline of quench-chase-pulse experiment in RbAp48 ON or OFF cells stably expressing SNAP-CENP-A. Bottom: representative images of G1 cells in which newly synthesized CENP-A was labelled with TMR-Star in RbAp48 ON or OFF cells. CENP-T was used as a centromere marker. Newly synthesized CENP-A were not detected in RbAp48 OFF cells. Bar, 10 μm. (d) Quantification of intensities by TMR-Star at indicated time points after tetracycline addition to RbAp48 conditional knockout cells. Five hundred centromeres in 100 different cells were quantified for each measurement. Error bars represent s.d. Asterisk indicates statistically significance (P<0.0001) by Student's t-test. (N=500). (e) Percentages of cells with diffused signals for newly synthesized SNAP-CENP-A are shown at indicated time points after tetracycline addition to RbAp48 conditional knockout cells. Definition of diffusion is in Supplementary Fig. 3D. (f) Western blot analysis with anti-CENP-A antibody in immunoprecipitates with anti-FLAG antibody in RbAp48 OFF cells expressing FLAG fused wild-type RbAp48 or Y32H mutant RbAp48. (g) Comparison of levels for H4K5ac or H4K12ac in RbAp48 OFF cells expressing FLAG fused wild-type RbAp48 with those in or Y32H mutant RbAp48.