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, Les cellules sont lavées avec du PBS (1X) et récupérées dans des tubes eppendorfs. Elles sont culotées par centrifugation pendant 5min à 2500rpm. Les cellules sont ensuite incubées dans la glace avec du tampon hypotonique, p.10
, les cellules sont lysées au dounce
, Afin de récupérer la fraction membranaire totale : Le surnageant est ultracentrifugé
, Afin de séparer les fractions endo-lysosomale et endosome/membrane plasmique : Le
, Les cellules sont culotées par centrifugation pendant 5min à 2500rpm. Les cellules sont lysées avec le tampon de lyse (0,34M saccharose, Les cellules sont lavées avec du PBS (1X) et récupérées dans des tubes eppendorfs
, 1mM DTT, 600mM KCl, 150mM NaCl, 150 mM pH 7
, ou non traitées) pendant une nuit avec la molécule d'IPA3 utilisé à une concentration finale de 20µM. Après l'incubation, les fractions cytoplasmique et membranaire sont séparées. La fraction membranaire est resolubilisée en tampon Tris HCl, Immunoprécipitation En utilisant l'épitope Flag: Les cellules sont traitées
, Sigma Aldrich M8823) pendant une nuit à 4°C sous rotation. Après plusieurs lavages en PBS les protéines sont éluées avec du tampon Laemmli sans agent réducteur, Chacune des fractions est incubée avec des billes magnétiques Anti-Flag M2
EDTA 0.5mM, NP-40 0.5% pendant 30 min dans la glace, puis centrifugées à 13200 rpm pendant 30 min. Le lysat cellulaire est ensuite incubé avec des billes magnétiques GFP-Trap (Chromotek) pendant une nuit à 4°C sur une roue, Les cellules sont lysées dans un tampon contenant du Tris 10mM pH 7.5, NaCl 150mM ,
, Production de protéines recombinantes en système bactérien et GST-Pull Down La production des protéines de fusion GST-PAKcrib et GST-Rhotekin est réalisée dans les 37°C jusqu'à l'obtention d'une DO de 0,6. La production de protéines recombinantes est induite par l'ajout d'IPTG à 1mM. Après une incubation d'une nuit à température ambiante sous agitation, la culture bactérienne est centrifugée à 5000rpm pendant 20min à 4°C
, En parallèle les cellules sont lysées dans un tampon contenant Tris-HCl 50mM pH 7
, MgCl 2 10mM, glycerol 5%, Na 3 VO 4 1mM. Les cellules sont centrifugées pendant 10 min à 13200 rpm à 4°C. Le lysat cellulaire était incubé pendant 1h avec du lysat bactérien contenant la protéine GST-PAKcrib ou la protéine GST-Rhotekin puis de nouveau 1h avec des billes glutathione-Sepharose. Après l'incubation, les billes sont collectées par centrifugation, NaCl, vol.100, p.1
, Triton X-100, 1mM dithiothréitol (DTT), 100mM NaCl et 30mM MgCl 2 . L'élution des protéines est réalisée avec du tampon Laemmli
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URL : https://hal.archives-ouvertes.fr/hal-01482390
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