1 / 1

METHODS Immunization procedures

METHODS Immunization procedures

arich
Télécharger la présentation

METHODS Immunization procedures

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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-Emonoclonal 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 Mad1were 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. Cell106(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 Rep3(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 J17(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 Cell15(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. Cell91(3): 357-366 Figure S3

More Related