In Vivo Ubiquitination Assay

The ubiquitination assay was performed as described by Choo et al. (41). The reaction mixture (40 μl) contained 8 μl of 5X ubiquitination buffer (100 mM Tris-HCl, pH 7.5, 25 mM MgCl₂, 2.5 mM DTT, 10 mM ATP), 250 ng of ubiquitination E1 (E-305, Boston Biochem, Inc), 500 ng of ubiquitination E2 (E2-656, Boston Biochem, Inc), 500 ng of ubiquitin (U-110, Boston Biochem, Inc), 500 ng of rPmTRIM50-like, 250 ng of VP19, VP24, VP26, or VP28. The mixture was incubated at 37°C for 3 h. The reaction was stopped by adding SDS-PAGE sample buffer and boiling for 10 min. Ubiquitination was detected by western blotting with anti-Ub (Boston Biochem, Inc.; 1:1000 in PBS) and HRP-conjugated goat anti-rabbit IgG (1:20000). The VP19, VP24, VP26, and VP28 proteins were detected by western blotting using HRP-conjugated GST tag mouse monoclonal antibody (Proteintech, Guangzhou, China; 1:20000 in PBS).