, All immobilisation steps were performed at a flow rate of 5 ml/ min with final mRNA concentration of 10 µg/ml. Total amount of immobilized ligand was about 1100-1500 RU. The channel (Fc1) was used as a reference surface for all non-specific binding measurements. For binding analysis, cytoplasmic lysates were injected first at 100 µg/ml over the immobilized surface for 2 min at a flow rate of 30 ml/min. Thereafter, the hnRNP H/F antibody was injected at a concentration of 200 µg/ml for 1 min and with the same flow rate settings. The binding of antibodies to molecules, 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20 (GE Healthcare)
, Eluates from chromatography experiments were loaded on a 6% UREA polyacrylamide gel and electrophoresed at 4°C for 1 h at 100 V in 0.5× TAE buffer, and then transferred to either a Biodyne B nylon membrane (Thermo Scientific, 77010) or Hybond-N + nylon membrane (Amersham Biosciences, RPN203B)
, DDX3X (1:1000, Santa Cruz sc-365768), LARP1 (1:1000, Bethyl A302-087A), hnRNP H/F (1:1000, Western Blot antibodies. For immunoblotting analysis, proteins were resolved on 12 or 7% denaturing polyacrylamide gels and were transferred to nitrocellulose membranes. The blots were blocked for 30 min with TBST-5% milk and then probed overnight with primary antibodies against DHX36 (1:1000, Abcam Ab70269), DHX9 (1:1000, Abcam Ab54593), vol.2, p.1000
, 1 mg/ml cycloheximide (CHX) for 15 min at 37°C, washed twice with ice-cold phosphate-buffered saline supplemented with 0.1 mg/ml CHX (PBS/CHX), and scraped on ice in PBS/CHX. After centrifugation for 5 min at 200 g, the cell pellet was gently resuspended in 450 ?l of hypotonic lysis buffer
, The settings were as follows: fraction time, 62 s/ fraction; chart speed, 60 cm/h; sensitivity of the OD 254 recorder, 0.5. The absorbance at 254 nm was measured continuously as a function of gradient depth; 16 fractions of approximately 0.8 ml were collected. The fractions recovered from the gradient were either analyzed individually or divided into three groups, fractions containing the most actively translated mRNAs, containing more than four ribosomes and called heavy polysomes (HP), fractions containing actively translated mRNAs containing two to three ribosomes, called light polysomes (LP) and fractions containing untranslated mRNAs (non-polysomes (NP)). Equal amounts of RNA from the NP, LP and HP fractions were extracted by using Trizol LS (Invitrogen), U/ml RNaseOUT (Invitrogen, 10777019), 0.1 mg/ml CHX and 10 µl/ml of Protease Cocktail Inhibitor (Sigma, P8340)). The lysate was vortexed for 5 s, incubated on ice for 5 min and 26 µl of 10 % Triton X-100 and 26 µl of 10% sodium deoxycholate were added
, Sigma P8833) for 10 min at 37°C. Cells were washed twice in ice-cold PBS, scrapped on ice in PBS and collected by centrifugation at 200 g for 5 min, Cell were lysed in 50 mM HEPES pH7.0, 150 mM NaCl, 10% Glycerol, 1% Triton, 10 mM Na 4 P 2 O 7 , 100 mM NaF, 1 mM EDTA et 1.5 mM MgCl 2 and 10 µl/ml Protease Cocktail Inhibitor (Sigma, P8340) buffer and puromycin incorporation was analyzed by western Blot
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, Acknowledgements We are grateful to S. Queille for assistance with immunofluorescence experiments; to M-J. Pillaire for discussion and materials; to A. Willis, for providing the hnRNP I antibody
, Moyal laboratories as well as N. Puget, D. Gomez and I. Gallouzi for discussions and advices. We thank C. Broussard (LC-MS supervision), M. Leduc (LIMS management), P. Mayeux (proteomic expertise and experimental design) from the 3P5 proteomic facility of the Université de Paris. The Orbitrap Fusion mass spectrometer was acquired with funds from the FEDER through the "Operational Programme for Competitiveness Factors and employment 2007-2013" and from the "Canceropole Ile de France
, Technology cluster of CRCT) for helping with surface plasmon resonance experiments and confocal imaging
, Olichon for helping with RIP assays using the BG4 antibody; A. De Magis for helping with immunofluorescence assays. This work was supported by institutional grants (from INSERM, Université Toulouse III -Paul Sabatier, CNRS) and by funding from LNCC (Ligue Nationale Contre le Cancer), ARC (Association pour la Recherche contre le Cancer), Emergence Cancéropole GSO, Laboratoire d'Excellence TOUCAN (ANR11-LABX) and ANR, MLB was supported by the Midi-Pyrénées Region/INSERM, PH by ANR
, performed most of the experiments, together with A.C., with assistance by L.D. and C.H. A.C. made all the figures. J.G. performed TCGA analysis. J.P.H. provided GBM samples. F.G. designed and supervised the proteomic analysis. A.A. performed proteomic analysis and statistical data treatment
, CD experiments. S.M. wrote the manuscript with input from A