FIG. 7.

Sumoylation of myocardin was differentially regulated by the small SUMO multigene family members. (A) Free myocardin and forms of myocardin sumoylated by Flag-tagged SUMO-1, -2, and -3 were detected by anti-V5 Western blotting (upper panel). Flag antibody and HA antibody were used to evaluate the equivalent expression levels of SUMO proteins (middle panel) or PIAS1 (lower panel), respectively, in various groups. Western blotting was conducted with extracts from HeLa cells transfected with V5-tagged myocardin alone or in combination with one of the Flag-tagged SUMO-encoding vectors in the presence or absence of HA epitoped PIAS1 as indicated. The asterisk denotes the SUMO-conjugated myocardin induced by PIAS1 (upper panel). (B) Reporter gene activity was analyzed for CV1 cells transfected with myocardin together with either SUMO-1, SUMO-2, or SUMO-3. Data obtained from two independent experiments are shown as percent activation relative to that for myocardin and SUMO-1, which is taken as 100-percent activation.