Protease inhibitor cocktails 1 and 2

Cells were harvested by scraping in radioimmunoprecipitation assay buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mm sodium molybdate, and 0.5% Nonidet P-40) containing protease inhibitor cocktails I and II (Sigma). Cell lysates were centrifuged at 16,000 × g for 10 min at 4°C. A portion of the supernatants was incubated sequentially at RT for 10 min with Dynabeads (Invitrogen) conjugated to anti-Myc antibody (Invitrogen), high affinity anti-HA antibody (Roche) or anti-M2 FLAG antibody (Sigma). Magnetic beads were washed three times with 200 μl of ice-cold PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4). Bound protein complexes and input fractions were examined by Western blot analysis using mouse-anti-Myc (Invitrogen), mouse anti-FLAG (Sigma), rat anti-HA (Roche), or mouse anti-ITCH (BD Transduction Laboratories) antibodies.

Protein was extracted with RIPA buffer (Thermo Fisher) supplemented with protease inhibitor cocktails I and II (Sigma) and phosphatase inhibitor cocktail set III (Calbiochem). Protein was quantified with Pierce BCA protein assay kit (Thermo Fisher) and analysed with a Sunrise Tecan plate reader. After Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electro‐transfer onto Polyvinyl‐Difluor membranes (GE Healthcare), Western blotting was performed with the following primary antibodies and concentrations: GAPDH (sc‐365062; Santa Cruz; 1:10,000), PAX3 (AB_528426; DSHB; 0.5 μg/mL), Twist2 (ab66031; Abcam; 1 μg/mL), IMP1/2/3 (sc‐271785; Santa Cruz; 1:1000), HMGA2 (no. 5269S; Cell Signalling; 1:1000), tubulin (ab210797; Abcam; 1:1000), p53 (sc‐126; Santa Cruz; 1:100), citrate synthase (G‐3; sc‐390693; Santa Cruz; 1:1000). Blots were stained with secondary antibodies conjugated with Horseradish Peroxidase, 1:2500, (Promega W401B, W402B) and visualized with ECL prime western blotting detection reagent kit (GE Healthcare).