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Materials and Methods
Antibodies
Other rabbit polyclonal antibodies used in this study were as follows: anti-Smc1, -Smc3, -SA1 and -SA2, all of which recognize both Xenopus and human proteins (Losada et al., 1998; Losada et al., 2000); anti-CAP-E/Smc2 (Kimura et al., 2001); anti-XCAP-E (Hirano and Mitchison, 1994); anti-XCAP-H (Hirano et al., 1997); anti-XCAP-H2 and Alexa 488-conjugated anti-XCAP-G (Ono et al., 2003); anti-XINC and anti-XAUB (MacCallum et al., 2002); anti-hCENP-E (a gift from T. J. Yen); anti-hOrc2 (a gift from B. Stillman). Mouse monoclonal antibodies against human aurora B (AIM-1; BD Biosciences) and a-tubulin (clone DM1A, Sigma) were also used.
References
Hirano, T. and Mitchison, T. J. (1994). A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell 79, 449-458.
Hirano, T., Kobayashi, R. and Hirano, M. (1997). Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89, 511-521.
Kimura, K., Cuvier, O. and Hirano, T. (2001). Chromosome condensation by a human condensin complex in Xenopus egg extracts. J Biol. Chem. 276, 5417-5420.
MacCallum, D. E., Losada, A., Kobayashi, R. and Hirano, T. (2002). ISWI remodeling complexes in Xenopus egg extracts: identification as major chromosomal components that are regulated by INCENP-aurora B. Mol. Biol. Cell 13, 25-39.
Files in this Data Supplement:
Fig. S1. Cell-cycle-regulated localization of Pds5A in HeLa cells. HeLa cells grown on coverslips were fixed with parformaldehyde and stained with an antibody against Pds5A (green) and DAPI (red). Merged images are also shown. Bar, 5 mm. Pds5A shows a behavior similar to that of cohesin: it is present inside the nucleus during interphase, but it is excluded from chromosomes from prophase to telophase, when it re-enters the daughter nuclei.
Fig. S2. An abnormally high level of cohesin is detectable in mitotic chromosomes assembled in Pds5-depleted Xenopus egg extracts. (A) HSS were immunodepleted with anti-Polo kinase (Plx1-dep, lane 6), a mixture of anti-INCENP and anti-aurora B antibodies (AUB-dep, lane 7), a mixture of anti-Pds5A and anti-Pds5B antibodies (Pds5-dep, lane 8), or control IgG (mock-dep, lanes 1-5). To estimate the efficiency of depletions, 1-ml aliquots of each extract were loaded on a gel alongside different amounts of the mock-depleted HSS (0.02, 0.05, 0.1, 0,2 and 1 ml [lanes 1-5]) and were analyzed by immunoblotting with the indicated antibodies. Depletions were >95% in all cases. (B) Sperm chromatin was incubated in the depleted interphase HSS for 2 hours and then each sample was split into two aliquots. One aliquot was processed for chromatin isolation without further incubation (lanes 2-5). The other was converted into mitosis by addition of a half volume of CSF HSS, incubated for another 2 hours, and processed for chromatin isolation (lanes 6-9). The chromatin fractions were analyzed by immunoblotting with the indicated antibodies. An aliquot of mock-depleted interphase HSS was analyzed in parallel (lane 1). In interphase, none of the depletions affected loading of cohesin (Scc1) on chromatin (lanes 2-5). Upon entry in mitosis, histone H3 became phosphorylated (H3P) in all except the aurora B-depleted sample, and condensin (CAP-E) was targeted independently of polo, aurora B and Pds5 (lanes 6-9). In contrast, dissociation of cohesin from chromatin was partially compromised when polo or aurora B was not present, as we had shown previously (Losada et al., 2002). A similar defect in cohesin’s release was also observed in the Pds5-depleted extract, further supporting the immunofluorescent data shown in Fig. 7B.
Fig. S3. Mitotic phosphorylation of Pds5B in Xenopus egg extracts. (A) Sperm chromatin was incubated in interphase HSS for 2 hours (I, lanes 1 and 3) and then one half of the sample was induced to enter mitosis by addition of a half volume of CSF HSS (M, lanes 2 and 4). Aliquots of the extracts (lanes 1 and 2) and the isolated chromatin fractions (lanes 3 and 4) were analyzed by immunoblotting with antibodies against topoisomerase II, Pds5A, Pds5B and Scc1. A sample from a mock assembly reaction without sperm chromatin (lane 5) was analyzed in parallel. Pds5B displayed a mitosis-specific mobility shift both in the soluble extract (lane 2) and in the chromatin fraction (lane 4). (B) To test whether the shift was under the control of Cdk-dependent phosphorylation, aliquots of an interphase HSS (I, lane 1) and a CSF HSS (M) either untreated (lane 2) or treated for 30 min with 0.8 mM of the Cdk inhibitor roscovitine (lane 3) were analyzed by immunoblotting. We found that the mitosis-specific mobility shift of Pds5B was in fact suppressed in the presence of roscovitine (upper panel). The same was true for the cohesin subunit SA1 and the condensin subunit CAP-H, both known to be phosphorylated in mitosis (Hirano et al., 1997; Losada et al., 2000). No change in the mobility of Scc1 was detected under this condition.
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