To verify whether UL44 could also be sumoylated in mammalian cells, we expressed UL44-FLAG and HA-SUMO-1 fusion proteins, in the presence of either wild-type Ubc9 or the catalytically impaired mutant Ubc9C93S in Phoenix cells and analyzed cell lysates by western blotting with anti-FLAG, anti-HA, and anti-Ubc9 antibodies. As expected, several bands corresponding to SUMO-1-conjugated proteins that reacted with an anti-HA antibody were detected upon co-expression of wild-type Ubc9, while less SUMO-1 conjugation was observed in the presence of the Ubc9C93S mutant (Fig. 3A) . This was not due to differences in expression of the two Ubc9 variants, as evidenced by western blot analysis with the anti-Ubc9 antibody (Fig. 3A) . When UL44-FLAG was expressed in the absence of HA-SUMO-1 and Ubc9, a single band with an apparent molecular mass of ,50 kDa was detected using the anti-FLAG antibody. In contrast, slower migrating bands similar to those observed in in vitro sumoylated products ( Fig. 2A) were observed upon co-expression of UL44-FLAG with HA-SUMO-1 and wild-type Ubc9 (Fig. 3A) . These bands were significantly reduced in the presence of the Ubc9C93S mutant (Fig. 3A) , demonstrating that they were dependent on the catalytic activity of Ubc9, with the residual slower migrating bands due to the activity of wild-type endogenous Ubc9.