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. Jean-philippe, , vol.3

, Fariba Nemati 2,6 , Didier Decaudin 2,6 , Guillaume Bataillon 7, Fatima Mechta-Grigoriou, vol.7, issue.7, p.13

U. Cnrs and I. Curie, , vol.26, p.75005

, Statistical Methods for Precision Medicine, INSERM U900

, -Bioinformatics and Computational Systems Biology of Cancer, Mines Paristech, INSERM U900, Institut Curie, PSL University, 26 rue d'Ulm, F-75248 Paris Cedex 05

(. D. -dna-repair-&-uveal-melanoma, I. Inserm-u830, and . Curie, , vol.26

, 26 rue d'Ulm, F-75248 Paris Cedex 05, France. 7-Department of pathology

, H+L) highly cross-adsorbed secondary antibody Alexa Fluor 568 (1/500, Invitrogen #A-11031), goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody Alexa Fluor 488 (1/500, Invitrogen #A-11008), vol.26, p.1200

, Cell lines Cell were fixed in cold methanol (-20°C) for 5 min, washed and permeabilized 3 times for 5 minutes using PBS-T (PBS 1X + 0, p.2

, Cells were incubated 1h at RT with primary antibodies diluted in PBT + 0.5% BSA. We used the same antibodies described above and cells were washed 3 times for 5 min and incubated 30 min with secondary antibodies diluted in PBT + 0.5% BSA: Goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody Alexa Fluor 568 (1/500, Invitrogen #A-11031), Goat anti-Rabbit IgG (H+L) highly cross-adsorbed secondary antibody Alexa Fluor 488 (1/500, Invitrogen #A-11008), Sodium Azide). Next, cells were blocked for 30 min at RT with PBS-T supplemented with 0.5% BSA

, Rac1 activation assay: Pull down assay: We performed Rac1-GTP pull-down assay using the Rac1 activation kit (# BK035-S, Cytoskeleton) according to the manufacturer's instructions. After centrinone or doxycycline treatment (and corresponding controls), adherent cells were scrapped and collected in lysis buffer. Then, protein extracts were incubated with PAK-PDB affinity beads. All the experiments were done at 4 °C. Next, beads were washed and resuspended in laemmli buffer for western blotting analysis. Western blotting: Proteins were separated on 4-20% SDS electrophoresis gel and transferred onto PVDF membranes using Trans-Blot Turbo Transfer System (#1704156, Biorad). Images were acquired using Chemidoc Imaging system (Biorad) and band intensities were quantified using Image, BSA and once with PBS 1X, then mounted with mounting medium home-made. found in 10 consecutive high-power fields (HPF)

, CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to Renata Basto (renata.basto@curie.fr) or Oumou Goundiam (oumou.goundiam@curie.fr), vol.REFERENCES, p.1

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