K63 linkage specific anti ub

E1 and E2 enzymes and Ub were either purified in house or purchased from Boston Biochem, UBPBio, or Enzo Life Sciences. Ubiquitination reactions were incubated at 37°C in 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), or 1 mM β-mercaptoethanol and typically contained E1 (50 or 100 nM), E2 (280 nM Ubc13/Uev1A, 1 μM Ubc13, or 1 μM Ubc5b), E3 (200 nM TRIM25 or 5 μM RING), Ub (40 μM), ATP (3 mM), and MgCl₂ (5 mM). Reactions were stopped by addition of SDS-PAGE sample buffer and boiling for 10 min. Typically, time points were taken at 0, 5, and/or 20 min. Immunoblots were performed with anti-Ub (1:2,000; P4D1; Santa Cruz Biotechnology), K63-linkage-specific anti-Ub (1:1,000; Enzo Life Sciences), anti-pentaHis (1:1,000; QIAGEN), and anti-FLAG M2 (1:5,000; Sigma). Signals were detected with a fluorescent secondary antibody (Rockland) and a LI-COR Odyssey scanner.