To identify the residues that are modified by SUMO1 we conducted arginine replacement mutagenesis of lysines on ASC and co-expressed each as a Flag-tagged construct with SUMO1 and UBC9 in HEK-293T cells, then assessed SUMOylation as previously described. As this experiment did not reveal a change in ASC SUMOylation despite separately mutating every lysine, and because the Flag epitope conforms to a SUMO motif (Supplementary Fig. 7a−c), we repeated the experiment with an HA-tagged ASC. These constructs identify that the lysine residues at positions 21 and 109 impacted ASC SUMOylation (Fig. 7a, b). Computer-aided prediction (by SUMOplot) distinguishes these same residues as putative SUMOylation sites (Fig. 7c). The lysine residue at position 109 is part of a flexible link between the proteins caspase and recruitment domain (CARD) and pyrin domain (PYD), while the lysine at position 21 forms a pocket with lysine residues number 22, 24 and 26 on the PYD of ASC (Fig. 7c)38. We exploited the Flag tag as an affixed SUMO site to try to distinguish how these two regions affected ASC SUMOylation. Mutation of the lysine
