in vitro Ubiquitination Assay Protocol

For in vitro ubiquitination assay, cells carrying plasmid ptifHis-ubi (expressing His₆-tagged ubiquitin under control of the tif51 promoter) were grown for 12 h and then treated with BZ for 12 h. Cells were then harvested and resuspended in lysing buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1% Nonidet P-40, 10 mM N-ethylmaleimide, 1 mM PMSF, and a complete protease inhibitor mixture) and lysed using a FastPrep-24 bead beater (MP Biomedical). The cell lysates were incubated with Ni-NTA agarose beads overnight at 4 °C. Beads were washed with washing buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.1% Nonidet P-40, 10 mM N-ethylmaleimide, 1 mM PMSF, and a complete protease inhibitor mixture), and bound proteins were eluted by boiling in SDS-PAGE sample buffer and were analyzed by Western blotting.