Trim25

According to the supplier’s instructions, RIPA lysis buffer (Beyotime) was used to isolate AML samples and cells’ total protein. The protein samples (20 µg) were separated using SDS-PAGE. Subsequently, the proteins were transferred to PVDF (Millipore, Billerica, MA). The membrane was blocked in 5% skim milk at room temperature for 2 h, and then incubated at 4°C with TRIM25 (Abcam) and β-actin (Abcam) overnight. After setting with HRP-linked secondary antibody at room temperature for 1 h, the protein bands were visualized using an electrochemiluminescence kit (Pierce Biotechnology), adopting β-actin protein as internal controls.

For immunoprecipitation, cells were thoroughly washed with PBS and lysed in total cell lysis buffer (20 mM Tris-Cl pH 7.4, 1% (v/v) IPEGAL^® CA-630, 150 mM NaCl), supplemented with 100 µM PMSF, 1 mM Na₃VO₄, 50 mM NaF, 2 mM N-ethylmaleimide (NEM), and cOmplete™ EDTA-free protease inhibitor cocktail tablets (Roche, Basel, Switzerland). Lysates were clarified by centrifugation at 16,100 rcf (13,200 rpm) for 15 min at 4 °C, then added to anti-FLAG M2 magnetic beads (Sigma-Aldrich, St. Louis, MI, USA) or anti-mCherry antibody (Abcam, Cambridge, UK #ab167453), anti-TRIM25 (Abcam, Cambridge, UK #ab86365) or anti-DDX3X (Biorbyt, Cambridge, UK #ORB167469) immobilised on Dynabead^® Protein G magnetic resin (Life Technologies, Carlsbad, CA, USA). Immunoprecipitation proceeded overnight at 4 °C. Captured proteins were extensively washed with total cell lysis buffer then with PBS and eluted by boiling in 2× Laemmli sample buffer.