DOI: 10.5281/zenodo.6795454
title: Trim71 partners after SETDB1 acute depletion

description: We immunoprecipitated the cytoplasmic GFP-tagged Trim71 (Trim71) and its specific partners in ESCs, where we acutely depleted Setdb1 after 48h of tamoxifen treatment (KO). In order to generate the Setdb1 cKO allele, exons 15 and 16, which encode the core amino acids of the catalytic domain, have been flanked with two lox-P sites recognized by Cre recombinase enzyme. Cre-oestrogen receptor fusion gene Mer-Cre-Mer has been introduced to induce acute Setdb1 KO after Tamoxifen treatment. Cells were lysed in buffer A (20 mM HEPES pH 7, 0.15 mM EDTA, 0.15 mM EGTA, 10 mM KCl), 10% NP40 and SR buffer (50 mM HEPES pH 7, 0.25 mM EDTA, 10 mM KCl, 70% (m/v) sucrose) with the addition of protease and phosphatase inhibitors (Sigma). Cell lysates were centrifuged at 2000 g for 5 min and the supernatant was recovered (cytoplasmic fraction) (as in (22)). Then high and low salt buffer were added (20 mM Tris-HCl pH 7.65; 0.2 mM EDTA; 25% glycerol; 900 mM NaCl; 1.5 mM MgCl2) to a final NaCl concentration of 300 mM to extract nuclear proteins. The nuclear extracts were next treated with Micrococcal nuclease (0.0125 U/ml) at 37°C during 10 min in addition to 1 mM CaCl2. EDTA was then added to 4 mM final concentration in order to stop the nuclease reaction. Finally, sonication was performed for 10 min (30 sec ON, 30 sec OFF) at medium frequency (Bioruptor Diagenode). The nuclear and cytoplasmic fractions were ultra-centrifugated at 40000 rpm for 30 min and pre-cleared with protein G-agarose beads (Sigma) during 2h at 4°C. Immunoprecipitations were carried out overnight at 4°C using 5 μg of each antibody. Ultralink A/G beads (Perbio) were blocked overnight at 4°C with 0,3 % BSA and 0.5 μg/μl ssDNA and then incubated with the immunocomplexes the next day for 2 h, at 4°C.<br> For immunoprecipitation using Myc-Trap technology, the Myc-Trap beads were equilibrated with wash buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.15 mM EDTA). Immunoprecipitations were carried out for 1 h or overnight at 4°C using 25 L of Myc-Trap bead slurry. The immunocomplexes were washed four times in wash buffer and the proteins were eluted in NuPAGE 4X loading buffer (Life Technologies) and 10X reducing agent at 96°C during 5 min. The samples were processed by mass spectrometry at the Taplin facility (as in Fritsch, L., Robin, P., Mathieu, J.R., Souidi, M., Hinaux, H., Rougeulle, C., Harel-Bellan, A., Ameyar-Zazoua, M. and Ait-Si-Ali, S. (2010) A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex. Molecular cell, 37, 46-56). .mzXML is a simplified mzXML file compatible with external application, .mzid is an mzIdentML file of the search results. 51460 84498 1 3306 GFP 51463 84502 2 3306 GFP 49436 82628 3 3306 GFP 49437 82629 3 3306 GFP Trim71 51465 84501 2 3306 GFP Trim71 51467 84497 1 3306 GFP Trim71 51462 84500 1 Tam 3306 GFP 51464 84504 2 Tam 3306 GFP 49438 82630 3 Tam 3306 GFP 49439 82631 3 Tam 3306 GFP Trim71 51461 84499 1 Tam 3306 GFP Trim71 51466 84503 2 Tam 3306 GFP Trim71
description: Work in Ait-Si-Ali's laboratory was supported by the Fondation pour la Recherche Medicale (FRM, « Equipe FRM » grant # DEQ20160334922); Association Française contre les Myopathies Telethon (AFM-Telethon, grant # 22480); Agence Nationale de la Recherche (ANR, « MuSIC » grant # ANR-17-CE12-0010-01), Université Paris Diderot and the "Who Am I?" Laboratory of Excellence, # ANR-11-LABX-0071, funded by the French Government through its "Investments for the Future" program, operated by the ANR under grant #ANR-11-IDEX-0005-01. R.R. has been supported by a DIM Biotherapies – Paris and LABEX "Who am I?" (Université de Paris—Université Paris Diderot) fellowships. P.C-T. has been supported by the Colombian Administrative Department of Science, Technology and Innovation (COLCIENCIAS-COLFUTURO); Universidad del Rosario (Becas para Apoyo para Estudiantes Doctorales 2017); Colombian Institute of Educational Credit and Technical Studies Abroad (ICETEX); French Government Agency Campus France (Eiffel Excellence Scholarship Program); Fondation ARC pour la Recherche sur le Cancer and LABEX "Who am I?" (Université de Paris—Université Paris Diderot).