des protéomes entiers des cellules endothéliales
Culture et différents traitements des HUVECCulture cellulaire : Les conditions de culture des HUVEC sont décrites dans la section Matériels et
MATERIEL ET METHODES
186
Stimulation au TNFα/IFNγ et à l’IL-1β : Les conditions de stimulation des cellules endothéliales avec le
TNFα/IFNγ, et l’IL-1β sont également détaillées dans l’article (Gautier, Mouton-Barbosa et al. 2012) (cf Résultats, Partie II-II).
Extinction de l’expression de l’IL-33 par siRNA : Deux transfections successives de 6h avec 50 nM d’un
pool de siRNA IL-33 ou de siRNA contrôle (ON-TARGETplus SMARTpool, Thermo Scientific/Dharmacon) ont été réalisées à 24h d’intervalle sur les HUVEC en présence d’oligofectamine (Invitrogen) dans un milieu de culture sans sérum OptiMEM (Invitrogen).
Stimulation à l’IL-3395-270 : Les HUVEC ont été stimulées avec 100 ng/mL de l’IL-3395-270 recombinante purifiée produite dans E.coli pendant 6h ou 24h dans un milieu de culture ECGM (Promocell).
Préparation des extraits protéiques : les cellules ont été lysées directement sur boite de culture par
ajout de 2% SDS puis ont été soniquées.
Gel 1D SDS-PAGE et analyse protéomique des protéomes entiers des cellules endothéliales
Les extraits protéiques issus de la lyse des cellules endothéliales après les différents traitements (stimulation par le TNFα/IFNγ, après stimulation avec l’IL-1β, après extinction de l’expression de l’IL- 33 par siRNA et après stimulation avec l’IL-3395-270) ont été traitées et analysés selon un même protocole (fractionnement sur gel 1D, digestion trypsique, analyse nanoLC-MS/MS sur un LTQ- Orbitrap Velos (Thermo Fisher Scientific) couplé à une nanoHPLC (U3000, Dionex), interrogation des banques de données, analyse quantitative sans marquage avec MFPaQ) décrit dans la section Matériels et Méthodes de l’article (Gautier, Mouton-Barbosa et al. 2012) (cf Résultats, Partie II-II).
187
LISTE DES PUBLICATIONS
1. Raoul Mazars, Anne Gonzalez-de-Peredo, Corinne Cayrol, Anne-Claire Lavigne, Jodi L. Vogel, Nathalie Ortega, Chrystelle Lacroix, Violette Gautier, Gaelle Huet, Aurélie Ray, Bernard Monsarrat, Thomas M. Kristie, Jean-Philippe Girard The THAP-Zinc Finger Protein THAP1 Associates with Coactivator HCF-1 and O-GlcNAc Transferase. A LINK BETWEEN DYT6 AND DYT3 DYSTONIAS. J Biol Chem. 2010 Apr 30;285(18):13364-71
2. Emma Lefrançais, Stephane Roga, Violette Gautier, Anne Gonzalez-de-Peredo, Bernard Monsarrat, Jean-Philippe Girard, Corinne Cayrol. IL-33 is processed into mature bioactive forms by neutrophil elastase and Cathepsin G . Proc Natl Acad Sci U S A. 2012 Jan
31;109(5):1673-8.
3. Violette Gautier, Emmanuelle Mouton-Barbosa, David Bouyssie, Nicolas Delcourt, Mathilde Beau, Jean-Philippe Girard, Corinne Cayrol, Odile Burlet-Schiltz, Bernard Monsarrat, and Anne Gonzalez de Peredo. Label-free Quantification and Shotgun Analysis of Complex Proteomes by One-dimensional SDS-AGE/NanoLC-MS - EVALUATION FOR THE LARGE SCALE ANALYSIS OF INFLAMMATORY HUMAN ENDOTHELIAL CELLS. Mol Cell Proteomics. 2012
Aug;11(8):527-39.
4. Sophie Mourgues, Violette Gautier, Joris Slingerland, Christine Bordier, Amandine Mourcet,
Fréderic Coin, Wim Vermeulen, Anne Gonzalez de Peredo, Bernard Monsarrat, Pierre-Olivier Mari, Giuseppina Giglia-Mari. ELL, a novel TFIIH partner is involved in transcription restart after DNA repair. En préparation
189
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