Ubiquitin e1

In vitro ubiquitination assays were performed as described previously (20 (link)). Ubiquitin E1 (UBE1), ubiquitin E2 (UbcH5a), and HA-ubiquitin were obtained from a commercial source (Boston Biochem). FLAG-ZIC2 was used as a substrate. Ubiquitin conjugation reactions were performed using a final protein concentration of E1 of 60 nM, E2 of 300 nM, E3 (K-Rta) of 100 nM, and ubiquitin of 10 μM in ubiquitin conjugation reaction buffer supplemented with an energy regeneration solution (Boston Biochem) for 2.5 h at 37°C.

In vitro ubiquitination assay was performed according to the manufacturer’s manual (Boston Biochem, Cambridge, MA, USA). Ubiquitin, E1, UBCH5A (Boston Biochem), VSV-N-FLAG, and TRIM41-HA or Del-RING-HA bound to the anti-HA resin (Sigma) were incubated at 30 °C in the ubiquitin assay reaction buffer (Boston Biochem) for 2 h. The anti-HA resin was washed with 1 M urea for 15 min to exclude potential binding of unanchored polyubiquitin. Then the resin was incubated with 45 µL of 0.5 mg/mL HA peptide to elute VSV-N protein. The eluates were subsequently analyzed by SDS-PAGE, followed by Western blotting.