Supplementary methods
Cell culture. HeLa cells were grown in DMEM containing 10% foetal calf serum. Cells
were arrested in S-phase by treating cells with 2 µg/ml aphidicolin for 18 to 24 hours. G1
cells were enriched and released from mitotic cells obtained by mitotic shake-off.
Antibody, protein and centrosome purification. Rabbit polyclonal antibodies against
the C-terminus of human C-Nap1were produced as previously described1. Other
antibodies used in this study were: mouse anti-centrin antibody (20H5; a gift from J.
Salisbury, Mayo Clinic, Rochester, MN); mouse anti- tubulin (GTU-88; Sigma, St
Louis, MO); rabbit anti-human pericentrin2; rabbit anti-GFP (a gift from J. Kahana,
UCSD, San Diego, CA); mouse anti-GFP (3E6; Molecular Probes, Carlsbad, CA). All
secondary antibodies used in immunofluorescence microscopy were from Molecular
Probes. Glutathione S-transferase-tagged fusion proteins of full-length Xic1 and 34Xic1
were prepared and purified as described3,4. Non-degradable Cyclin B (90) and the Nterminal fragment (13-110) of sea urchin cyclin B containing a wild-type or mutated
destruction box were made as previously described5,6. Recombinant securindm was
purified from E. coli as described7. Centrosomes were isolated from HeLa cells as
described8.
Xenopus extracts. Xenopus CSF egg extracts were prepared as described9. CSF-released
was induced by adding CaCl2 to 0.4 mM final concentration. Spindle morphology and
sister-chromatid separation were examined as described7. CSF-released extract was
arrested at anaphase by the addition of non-degradable cyclin B (anaphase extract) as
described6,10. Interphase and cycling extracts9 were made from eggs activated with
calcium ionophore A23187. Interphase-arrested extract was made by treating cycling
extract with cycloheximide9.
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