HCMV UL44 SUMOylation Regulation

293 T cells were co-transfected with pCMV-Myc-UL44 or pCMV-Myc-UL44-K410R, pcDNA-His-PIAS3 (wild-type or C334S mutant) and other SUMOylation-related plasmids (pRK-Flag-SUMO-1/-2/-3, pcDNA-HA-UBC9) as indicated using Lipofectamine 2000 (Invitrogen). After 48 h, the in vivo SUMO modifications of UL44 and its K410R mutant in transfected cells were analyzed by Western blot using anti-Myc and anti-Flag antibodies, as described in our previous work [35 (link)]. Meanwhile, to analyze the SUMOylation of UL44 under HCMV infection, U251 cells were first transfected with siControl or siPIAS3, and 24 h later infected with wt-HCMV or v-K410R at an MOI of 1. At 48 hpi (hour post infection), total cell lysates were immunoprecipitated with anti-UL44 antibody or analyzed directly by SDS-PAGE, followed by detection of UL44 and UL44-SUMO proteins using anti-UL44 or anti-SUMO-1 antibody. Additionally, in vitro SUMOylation assays were performed with the SUMOylation Kit from Enzo Life Science (Catalog #BML-UW8955) according to the manufacturer’s suggestions.