As the lysine residues at positions 21 and 22 of ASC have been identified as being ubiquitinated and because it has been speculated that SUMOylation may function to stabilise proteins by counteracting ubiquitin-mediated degradation39,40, we sought to assess competition for these four lysine residues by SUMO1 or ubiquitin (Ub). Towards this, the WT or mutant ASC (4KR) was expressed in HEK-293T cells with ZBTB16, UBC9 and Ub. Immunoblotting of immunoprecipitated WT and 4KR ASC with an anti-Ub antibody detected an equivalent signal (Fig. 7f). This appears to dismiss the notion of competition for the lysine residues between positions 21 and 26 by the ubiquitination complex and is in keeping with the preceding measures that show no effect of ZBTB16 on the levels of ASC (Figs. 2c, d, 4e, 5e and Supplementary Fig. 2a, b, 4, 5b, c with 7g).
