Trim71 partners after SETDB1 acute depletion

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.
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).

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