Milea Timbergen

97 glycerol (Pierce IP lysis buffer) containing Halt Protease and Phosphatase inhibitor single- use cocktail (ThermoFisher Scientific). Wells were cleaned by scraping and the cell lysates collected and centrifuged at 11.000 x g for 10 minutes at 4°C to pellet insoluble cell debris. From the cleared lysates 10% was used as input control which is prepared for SDS-PAGE by adding an equal volume of 2 x Laemmli sample buffer with 0.1 M DTT (Laemmli/ DTT). FLAG-tagged β-catenin is immunoprecipitated from the remainder of the lysates using prewashed ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich, cat. No. A2220) for 2 h at 4°C. FLAG-beads were washed with lysis buffer for three times and resuspended in Laemmli/DTT. Input and IP samples were heated to 95°C for 5 minutes and subjected to SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in TBS/0.1% Tween’20 supplemented with 5% (w/v) BSA and incubated overnight at 4°C with rabbit monoclonal anti-DNMT1 (1:1000 DNMT1 XP®, D63A6, Cell Signaling Technology), mouse monoclonal anti-FLAG® M2 antibody (1:1000 Sigma-Aldrich, cat no. F1804) or mouse monoclonal anti-β-actin (1:10.000, Sigma-Aldrich, cat no. A5441). The primary antibodies were diluted in blocking buffer. HRP conjugated goat-anti-rabbit, goat-anti-mouse were used as secondary antibodies in TBS/0.1% Tween’20 supplemented with 5% (w/v) non-fat dried milk. Enhanced chemiluminescence (SuperSignal™West Pico Plus Chemilumininescent Substrate, Thermo Scientific) was used to visualize the bound antibodies in a ChemiDoc MP Imager (Bio-Rad). Ethical approval This study was part of a protocol entitled “Translational research on soft tissue sarcomas” which was assessed by the Medical Ethics Committee of the Erasmus MC, Rotterdam, the Netherlands (MEC-2016-213). Results Clinical characteristics of the patients included in the MeD-Seq analysis The vast majority of DTF tumours contain mutations in exon 3 of the CTNNB1 (β-catenin) gene. Interestingly, the mutations are almost exclusively confined to residues T41 and S45 preventing the phosphorylation of these residues and consequently stabilizing CTNNB1 and activating Wnt/β-catenin signalling. Although having similar effects, the T41A and S45F mutated DTF tumours were reported to display a different clinical behaviour for which the underlying biological mechanism is still unclear. Epigenetic alterations may be important in this respect, 4