Creatine phosphate

To produce the K63-linked polyubiquitin chains, 5 μM Ubiquitin (#E1100, UBPBio), 50 nM UBE1 (# B1100, UBPBio), 200 nM Ubc13, 200 nM Mms2 and energy regenerating source (1 mM ATP (Roche), 10 mM creatine phosphate (# 030-04584, Wako), and 20 μg/ml creatine kinase (# 10127566001, Roche)) were mixed in buffer 5 (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 10% Glycerol) at 32 °C for 3.5 h. The K63-linked polyubiquitin chain product or purchased ubiquitin (#D2300, UBPBio) were incubated with the purified hRQT complex-conjugating anti-FLAG® M2 Magnetic Beads (#M8823-5ML, SIGMA) in buffer 6 (50 mM Tris pH 7.5, 100 mM NaCl, 2.5 mM MgCl₂, 10 μM ZnCl₂, 10% Glycerol, 1 mM DTT, 0.1% NP-40, 100 mM l-arginine, 1 mM PMSF) at 23 °C for 15 min. To prevent non-specific binding, beads were washed five times and then eluted by 1 x Sample Buffer. In the analysis using purchased tetraubiquitin chains (Ubiquitin #E1100, K48-Ub4 #D1300, K63-Ub4 #D2300, UBPBio), 1.5 μg of mono- or K48-tetraubiquitin, 125 ng of K63-tetraubiquitin were incubated with the purified hRQT complex-conjugating anti-FLAG® M2 Magnetic Beads as described above.

Chemicals used in this study were mainly purchased from Nacalai tesque (Kyoto, Japan). Lysine, 3-phospho-D-glyceric acid barium salt dihydrate, folinic acid, and CTP were purchased from Tokyo Chemical Industry (Tokyo, Japan). Potassium glutamate, adenosine, and cysteine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Creatine kinase was purchased from Roche (Basel, Switzerland). ATP, GTP, NAD⁺, and creatine phosphate were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Tryptone and Yeast Extract were purchased from BD (Franklin Lakes, NJ, USA). UTP was purchased from Affymetrix (Santa Clara, CA, USA).