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 nuclear speckles evident for the heterogeneous pairings and a single cytosolic speck produced by ASC oligomerization (Fig. 6e). Measures of the total level of Venus fluorescence in cells transfected with the different ZBTB16 constructs and either ASC or UBC9 confirm their association and distinguish that they separately associate with the BTB and repressor domains of ZBTB16, respectively (Fig. 6f). Fluorescence produced by an association between ZBTB16 and the N-terminally tagged SUMO1 (nSUMO1) was partly retained with the carboxyl-terminally tagged SUMO1 (SUMO1c), thereby identifying that ZBTB16 can colocalize with SUMO1 other than as a substrate37. Importantly, although the colocalization between ZBTB16 and ASC or UBC9 produced low levels of Venus fluorescence compared to that of ASC and UBC9 or SUMO1, the expression of ZBTB16 significantly increased the level of fluorescence produced between tagged ASC and SUMO1, thereby supporting a positive function of ZBTB16 in the association of ASC with SUMO1 (Fig. 6g).
