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, Annexe n°1 : DDR1 contrôle la formation des invadosomes linéaires via la voie
, Cnn2 Calponin-2 yes
, Cnn3 Calponin yes
, Myef2 Myelin expression factor 2 ND yes Chung et al, Int J Clin Exp Pathol, vol.10, issue.4, pp.4682-4687, 2017.
, Myh9 Myosin, vol.9
, Annexe n°5 : Matériel et méthodes projet en cours n°2
, Modified Eagle Medium ; 4,5g/L D-glucose et pyruvate Gibco®) complémenté avec 10% de sérum de veau foetal et 1% d'antibiotiques (Pénicilline/Streptomycine). Ces lignées sont cultivées à 37°C, Les lignées cellulaires Huh7 et MDA-MB-231 (ATCC) sont cultivées dans un milieu DMEM GlutaMAX-I (Dubelcco's
, Les cellules Huh7 sont ensemencées en plaque 6 puits, puis transduites après 24 heures avec une gamme de multiplicité d'infection de 2-5-10, soit avec des plasmides lentiviraux contenant les différentes isoformes de DDR1 couplées à la GFP, soit avec un plasmide contrôle codant la GFP seule
, Les cellules sont ensemencées sur des boîtes de Pétri afin d'avoir au moins 80% de confluence 24 heures après ensemencement. La lyse est effectuée avec le tampon de lyse RIPA, 1% SDS, 1% NP40, 150mM NaCl, 1% sodium deoxycholate, 25mM Tris HCl pH 7,4) supplémenté des inhibiteurs de protéases (Complete Mini EDTA free 1X, Roche) et des inhibiteurs de phosphatases
, g à 4°C et le surnageant contenant les protéines est conservé. Les protéines sont dosées selon la méthode de Lowry (kit DC? Protein Assay, Biorad) au spectrophotomètre à 750nm
, Quarante ?g de protéines sont repris dans 5?L de tampon Laemmli
, Les protéines sont ensuite dénaturées à 95°C pendant 5 minutes. Les échantillons sont déposés dans un gel d'acrylamide 10% Biorad (kit fast cast Biorad). La migration est réalisée dans un tampon de migration 1X, concentration finale 1X)
, Glycine 28,8 g/l ; SDS 0,1%) à 100V, jusqu'à la sortie du front de migration du gel
, Les protéines sont ensuite transférées sur une membrane de nitrocellulose à l'aide du Transblot Turbo de BioRad (programme de transfert 10 minutes, 25 Volts, 1,3 Ampère). Les protéines totales sont révélées au rouge Ponceau afin de s'assurer de l'efficacité du transfert. La membrane est saturée dans du Blocking Buffer Odyssey, une heure, à température ambiante, Annexe n°6 : ASS1 : un marqueur des adénomes hépatocellulaires inclassés et à fort risque de saignements