6b, c). To detect if these proteins interact in situ at endogenous levels, we performed fluorescent proximity ligation assays (PLA) for the association of ASC with ZBTB16, SUMO1 or NLRP3 in BMDM cells that were untreated or treated with LPS alone or in combination with nigericin. A fluorescence signal was generated between antibodies for ASC and ZBTB16 in the nucleus of resting WT BMDMs, but not the Zbtb16-/- cells, which increased after immune stimulation (Fig. 6d). This protocol also confirmed the previously detected immune-mediated association between ASC and SUMO1 (Figs. 5f and 6d). Critically, this signal was heightened in the WT compared with the Zbtb16-/- BMDM, thereby supporting a positive role for ZBTB16 in ASC SUMOylation (Fig. 6d). ZBTB16 was also shown to promote the assembly of the inflammasome (shown in Fig. 4), as apparent from the heightened association between ASC and NLRP3 upon immune stimulation in the WT compared to the Zbtb16-/- BMDM (Fig. 6d). This assay confirms that ZBTB16 colocalizes with ASC to increase its colocalization with SUMO1 in the nucleus and subsequently promotes an association with NLRP3 in the cytosol.
