We next examined the effect of SUMOylation on ASC oligomerization. This was initially measured with differently tagged ASC constructs in HEK-293T cells. The indirect capture of a Flag-tagged ASC by immune-enrichment of an HA-ASC construct was increased by co-expressing SUMO1 with UBC9, as detected by immunoblot of immunoprecipitates (Fig. 8a). Involvement of the lysine residues at positions 21 and 109 in the association between separate ASC proteins was confirmed in the same manner using arginine replacement mutants (Fig. 8b). As an alternative measure, the multimerization of ASC was visualised using constructs tagged with either half of the split-Venus fluorophore. In this instance, a positive effect of SUMOylation on ASC oligomerization was demonstrated by showing that an inhibitor of SUMOylation (2-D08) reduced Venus fluorescence in the cytosol of cells (Fig. 8c). The lysine residues at positions 21 and 109 were also tested for their effect on ASC multimerization upon immune stimulation by expressing the WT or lysine mutant constructs in Asc-/- BMDMs. Immunofluorescent detection of ASC with an anti-ASC antibody shows the two lysines are important for the formation of ASC
