To assess if ZBTB16 might directly control the modification of ASC we assessed the association between the proteins. Measures of the localisation of the SUMO factors were carried out. Immunohistochemical labelling of BMDM using antibodies against ASC, SUMO1, the SUMO ligase UBC9, ZBTB16 and PML reveals that the proteins predominantly localise in the nucleus of unstimulated cells (Fig. 5f and Supplementary Fig. 6). Analysis of the expression of ASC and ZBTB16 detected by immunohistochemical double-labelling of BMDM transformed with a ZBTB16 expressing construct detects a degree of colocalization of ZBTB16 and ASC in the nucleus in resting cells. The corresponding reconstructed renderings by Imaris showed the contact area of ZBTB16 with ASC (5.45 μm2) (Fig. 6a and Supplementary Fig. 6). An association between ASC and ZBTB16 was supported by the co-immunoprecipitation of the proteins when overexpressed in HEK-293T cells (Fig. 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
