Identification of NEDD8 conjugation

We identified NEDD8 conjugation, as previously described.⁴³ After transfected with the plasmid for His₆-NEDD8 or His₆-NEDD8ΔGG mutant, cells were sub-cultured in two dishes. The cells in one dish were lysed for western blotting to confirm the expression level of input proteins, while those in the other dish were lysed in a denaturing buffer (6 M guanidine hydrochloride, 0.1 M Na₂HPO₄/NaH₂PO₄, 0.01 M Tris-Cl (pH 8), 10 mM imidazole, and 10 mM β-mercaptoethanol). The lysates were incubated with Ni²⁺-NTA-agarose beads (Qiagen) at room temperature for 4 h. To remove non-covalently bound proteins, the beads were washed in a denaturing buffer (8 M urea, 0.1 M Na₂HPO₄/NaH₂PO₄, 0.01 M Tris-Cl (pH 8), 20 mM imidazole, 10 mM β-mercaptoethanol, and 0.2% Triton X-100) at pH 8.0 for 5 min and done again in the buffer at pH 6.3 for 5 min. Then, His-tagged proteins bound to Ni²⁺ were eluted in a 2 × SDS denaturing buffer and subjected to western blotting.