1. METHODS Immunization procedures The Xenopus anti-Aurora B and anti-Mad2 antibodies have been described elsewhere (Castro et al, 2002; Lorca et al, 1998). Antibodies against the N-terminal domain of Xenopus Mps1 (residues 1 to 626) were obtained as previously described (Abrieu et al, 2001). CENP-E was detected by using anti-human CENP-E monoclonal antibody in Hela cells (1H1, AbCam) (Figures 4H and 5F) or anti-tail Xenopus CENP-E polyclonal antibodies inCSF extrats (Figures 1A, 3A, 5B and Figure S2) (Wood et al, 1997). Anti Xenopus Bub1 antibodies were generated against a GST fusion protein corresponding to the N-terminal domain of this protein (residues from 1 to 850). Anti Xenopus Rsk2 antibodies were obtained against either a His-tagged Xenopus Rsk2 protein (Full Length Rsk2 antibodies) or against a peptide corresponding to the 12 last C-terminal aminoacids (Cterminal Rsk2 antibodies). Finally, rabbit polyclonal antibodies anti-Xenopus Bub3 and Mad1 were generated against two peptides (MNTQTDMTGSNE and MDDSEDNTTVIS respectively) corresponding to the N-terminal sequence of these proteins. Anti-Cterminal human Rsk1 polyclonal antibody, anti-Cterminal human Rsk2 monoclonal antibody (E1), anti-Mad2 monoclonal antibody (17D10) and anti-Mad1 monoclonal antibody (9B10) were obtained from Santa Cruz Biotechnology. Anti-Cterminal Xenopus Rsk2 as well as anti-Cterminal human Rsk1 (Santa Cruz) were used in CSF extracts for immunoprecipitation, immunobloting as well as for immunofluorescence except for Figure 1A in which anti-full length Xenopus Rsk2 was also used. Plasmids pFastBac1-Xenopus Rsk2 wild-type and kinase dead mutant (K97R) constructs were kindly provided by Dr. JE Ferrell. Both cDNAs were subcloned in a pCS2 plasmid in which the GFP cDNA from the phmGFP vector (Promega) was subcloned in the StuI-XbaI site. Cell culture, transfection and immunofluorescence Human HeLa cells were grown in DMEM medium with 10% FBS and penicillin/streptomycin. Plasmid transfections were performed by using Fugene (Roche). For immunofluorescence, cells were first fixed with either 3,7% formaldehyde or with 100% methanol (when anti-INCENP or anti-human CENP- E antibodies were used) for 10 minutes at room temperature, permeabilized with 0,2% of Triton X100 in PBS for 10 min (except for anti-human Mad2 and Mad1 antibodies where we used 0,1% Triton W100) and blocked with 5% of FBS in PBS for 30 min. Polyclonal anti-XCENP-E tail (1/600), anti-XMad2 (1/200), anti-XBub1 (1/200) anti-XINCENP (1/400) (Vigneron et al, 2004) and monoclonal anti-hCENP-A (1/300, Abcam) and anti-hBubR1(1/500, BD Transduction Laboratories) were used as primary antibodies. The corresponding Alexa488 (1/600) or Alexa555 (1/1000) anti-rabbit or anti-mouse secondary antibodies (Molecular Probes) were used as indicated. References 1-Abrieu A, Magnaghi-Jaulin L, Kahana JA, Peter M, Castro A, Vigneron S, Lorca T, Cleveland DW, Labbe JC (2001) Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. Cell 106(1): 83-93. 2-Castro A, Arlot-Bonnemains Y, Vigneron S, Labbe JC, Prigent C, Lorca T (2002) APC/Fizzy-Related targets Aurora-A kinase for proteolysis. EMBO Rep 3(5): 457-462. 3-Lorca T, Castro A, Martinez AM, Vigneron S, Morin N, Sigrist S, Lehner C, Doree M, Labbe JC (1998) Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts. Embo J 17(13): 3565-3575 4-Vigneron S, Prieto S, Bernis C, Labbe JC, Castro A, Lorca T (2004) Kinetochore localization of spindle checkpoint proteins: who controls whom? Mol Biol Cell 15(10): 4584-4596 5-Wood KW, Sakowicz R, Goldstein LS, Cleveland DW (1997) CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment. Cell 91(3): 357-366 Figure S3