We next sought to map the interacting domains of the protein partners. For this, we used a bimolecular fluorescence complementation assay. This assay evaluates protein associations by tagging partners with the separate amino- or carboxyl-terminal fragments of a split-fluorophore. An association between the separate protein partners reconstitutes the full-length fluorophore and so is detected as a fluorescent signal35,36. ZBTB16, ASC, UBC9 and SUMO1 were differently tagged with the separate halves of split-Venus (coded V1 and V2) and co-expressed in HEK-293 cells. As well as the full-length ZBTB16, truncated constructs encoding the proteins’ Broad-Complex, Tram-track and Bric-a-brac (BTB) domain alone and this with the adjacent repressor domain (BTB-RD) were also tested. In addition, SUMO1 was alternatively tagged at either terminus to distinguish merely an association from covalent conjugation, based upon amino-tagging leaving free while carboxyl-tagging blocks the carboxyl diglycine residues required for SUMOylation. The pattern of Venus fluorescence produced by the oligomerization of ASC and its association with UBC9 or SUMO1 in HEK293 cells is shown (Fig. 6e). Two distinct patterns of fluorescence were produced by the ASC associations, with multiple
