Previous reports identified that the E3 SUMO ligase PML, which co-locates with ZBTB16, regulates ASC nuclear location31,32. As PML-nuclear bodies are SUMOylation hotspots, with ZBTB16 itself a SUMO substrate, we tested if ASC was modified with SUMO. To directly assess the SUMOylation of ASC, we transfected HEK-293T cells with ASC, the SUMO ligase UBC9 and His-tagged constructs of SUMO1-3 then enriched SUMO and its conjugates by binding to Nickel-NTA agarose resin (Ni-NTA) as described previously33. This identified that ASC is modified by SUMO1 (Fig. 5a and Supplementary Fig. 5a). Fittingly, this SUMOylation of ASC was reduced by either treatment with a pharmacological inhibitor (2-D08) or co-expression of the SUMO1-specific protease Sentrin-specific protease 1 (SENP1) (Fig. 5b, c)34. To evaluate the role of ZBTB16 in ASC SUMOylation, we overexpressed ASC, His-SUMO1 and UBC9 with ZBTB16 in HEK-293T cells. Immunoblotting of Ni-NTA enriched cell lysates detects ZBTB16 expression increased ASC SUMOylation (Fig. 5d). This ZBTB16-dependent increased ASC SUMOylation was confirmed for the endogenous proteins by immunoblotting peptides enriched with an anti-ASC or control anti-IgG antibodies from WT and Zbtb16- /- BMDMs with
