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 residues between positions 21 to 26 (coded 4KR) reduced the SUMOylation of the Flag-ASC construct (Supplementary Fig. 7d, e). This curtailed SUMOylation, even at extrinsic sites in the Flag epitope, suggests that the residues between 21 and 26 affect the recruitment of ASC to the SUMOylation complex. In keeping with this, mutating the lysine residues 21−26 affected ASC’s association with ZBTB16, as assessed by immunoblot analysis of WT and the 4KR mutant ASC proteins overexpressed and immunoprecipitated with ZBTB16 in HEK-293T cells (Fig. 7d). Accordingly, this mutation also ablated the ZBTB16-dependent increase in ASC SUMOylation (Fig. 7e).
