His-Tag Pull-Down Assay

pET-28a-UL44 was transformed into E. coli BL21(DE3) and induced by IPTG to express His-UL44 fusion protein. pCMV-Myc-UBC9 was transfected into 293T cells, the mammalian cell lysates were prepared after 48 h and used as the source of Myc-tagged UBC9 protein. His-tag pull-down experiments were performed using Pierce His Protein Interaction Pull-Down Kit following the manufacturer’s protocol (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche), and 10 mM β-mercaptoethanol was added into both lysis and elution buffer to prevent the aggregation of His-UL44. The bound proteins were analyzed by western blot using anti-His and anti-Myc antibodies (Sigma-Aldrich).