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1 Supplementary Figure S1 Figure S1. CENP- FP constructs target to centromeres irrespective of site of tagging. FP- CENP and CENP- FP constructs were transfected into U2OS cells and counterstained with anti- centromere antibody (ACA) in the appropriate color channel. A) EGFP- CENP- S; B) CENP- S- EGFP; C) CENP- S- mch; D) mch- CENP- S; E) EGFP- CENP- X; F) CENP- X- mch. Scale bar equals 5 microns.
2 Supplementary Figure S2 Figure S2. FCCS analysis of CENP- T and CENP- S. EGFP- CENP- T and mch- CENP- S were co- expressed and analyzed as in Figure 1. Correlation spectra for EGFP- CENP- T (green) and mch- CENP- S (red) show no cross- correlation (black).
3 Supplementary Figure S3 Figure S3. CLIP- 505 labeling of CENP- S- CLIP. Asynchronous cells were fixed and immunostained for CENP- A (green in merge). Cells were also labelled with CLIP- 505 to reveal CENP- S localization (red in merge). DNA was stained with DAPI (blue). Scale bar equals 10 microns.
4 Supplementary Figure S4 Figure S4. Short term FRAP with EGFP- CENP- S. Short- term FRAP with EGFP- CENP- S at different cell cycle stages. HeLa cells expressing mrfp- PCNA were transiently transfected with EGFP- CENP- S and cells were staged on the basis of PCNA pattern and nuclear size. At each stage, the indicated number of kinetochores were examined in at least 3 cells. Data are reported as the mean with standard deviations. No significant recovery in a 360 second time course was observed.
5 Supplementary Figure S5 Figure S5. Example of long- term CENP- S- EGFP FRAP recovery at centromeres. FRAP analysis in a G1 cell is presented. Two small fields are shown at each of a series of time points, containing equivalent kinetochore numbers. The upper field was bleached at the indicated time and images were collected at consecutive 30 minute intervals. The lower field is a control, unbleached region. Recovery approaches 100% in the bleached zone.
6 Supplementary Figure S6 Figure S6. FRAP with CENP- X. HeLa cells expressing mrfp- PCNA were transiently transfected with EGFP- CENP- X and cells were staged in S- phase on the basis of PCNA pattern and nuclear size. FRAP was performed as described on the indicated number of kinetochores. CENP- X recovery was rapid and approached 100%.
7 Supplementary Table 1: Primers used Primer Set Sense Primer, Antisense Primer, Product CENP-A RT CGCTTCCTCCCATCAACAC GGGCAGCTTCCTTATCAAGAG qpcr product CENP-B RT GAAGCCCAGTGCCCTACTC TCCTCATCATCATCGTCGTCTT qpcr product CENP-C RT AGTGAATCCAGTCCCATTGTTAG TCATCCTCTATCAACTTCGTATCAT qpcr product CENP-S RT CGCAGTGATGGAGGAGGAG CGCAAAGACAACCCACAGTAT qpcr product CENP-X RT CCTCCTGGCCACATTCCTG GATTTATTGATGTTGCTTTGTGAGAA qpcr product Cyclin A RT ACAGCCAGACATCACTAACAG CAGCACTGACATGGAAGACA qpcr product Cyclin B RT AGCAAGCAGTCAGACCAAAAT CAGTCAATTAGGATGGCTCTCA qpcr product CENP-R CENP-S CENP-T CENP-X CENP-S D AAACCTGTATTTTCAGGGCGCCACCATGGGCAT GCCTGTTAAAAGATCACTGAA AAACCTGTATTTTCAGGGCGCCACCATGGAGGA GGAGGCGGAGAC AAACCTGTATTTTCAGGGCGCCACCATGGCTGA CCACAACCCTGAC AAACCTGTATTTTCAGGGCGCCACCATGGAGGG AGCAGGAGCTGGAT AAACCTGTATTTTCAGGGCGCCACCATGGAGGA GGAGGCGGAGAC GGGGACCACTTTGTACAAGAAAGCTGGGTG TTTAAAATGGCTTTAAGGAATTCA GGGGACCACTTTGTACAAGAAAGCTGGGT ATTCTCACTTTCCACCACTCCAGC GGGGACCACTTTGTACAAGAAAGCTGGGTC TGGGCAGGGAAGACAGAGTT GGGGACCACTTTGTACAAGAAAGCTGGGTG AAGTCCAGGAGCAGCTGCG GGGGACCACTTTGTACAAGAAAGCTGGGTT GAATTACTCCTCCTGGCTAAGAGC Full length CDS Full length CDS Full length CDS Full length CDS N terminal HFD of CENP-S Supplementary Table 2: Antibodies used. Reactivity Species Conjugation IF WB Supplier CENP-A Mouse IgG 1/300 1/1000 Abcam CENP-B Mouse IgG - 1/250 W.Earnshaw CENP-S Rabbit IgG - 1/500 W.Wang CENP-X Rabbit IgG - 1/500 W.Wang CENP-T Rabbit IgG - 1/1000 Bethyl Labs CENP-W Rabbit IgG - 1/1000 Abcam Cyclin B Mouse IgG - 1/2000 Upstate Histone H3S10P Rabbit IgG - 1/2000 Millipore PCNA Human IgG 1/100 - KF.Sullivan Zwint Rabbit IgG 1/100 - Bethyl Secondary antibodies: Anti-Rabbit IgG Donkey Cy5 1/200 - Jackson Labs Anti-Mouse IgG Goat TRITC 1/100 - Jackson Labs Anti-Human IgG Donkey AMCA 1/100 - Jackson Labs Anti-Rabbit IgG Goat HRP - 1/10,000 Jackson Labs Anti-Mouse IgG Goat HRP - 1/10,000 Jackson Labs
8 Supplementary Table 3: Summary of F2H results RFP \ CENP-A CENP-M CENP-R CENP-S CENP-T GFP CENP-A CENP-M CENP-R CENP-S CENP-T denotes strong interaction + denotes detectable interaction +- denotes weak interaction - denotes undetectable interaction
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