To analyze UL44 sumoylation in transient expression assays, HeLa or Phoenix cells were transfected for 48 h with appropriate plasmids using the calcium phosphate precipitation method. For analysis of UL44 sumoylation during HCMV replication, HFF, U373-Neo, and U373-SUMO-1 cells were mock-infected or infected with HCMV at a multiplicity of infection (MOI) of 5 PFU/cell. Cells were harvested at different time points after infection and analyzed by western blotting as described below.