As ASC oligomerization induces the processing of the Caspase-1 zymogen, we measured its activity. A fluorescent Caspase-1 assay (FLICA29) detected heightened Caspase-1 activity in the WT compared to the Zbtb16- /- BMDMs treated with LPS and nigericin (Fig. 4c). The subsequent processing of cytokines by active Caspase-1 requires their recruitment to the inflammasome. We visualised the recruitment of a pro-IL-1β construct that was tagged at its termini with fluorophores as described previously30. This shows the increased aggregation of IL-1β into cytosolic puncta in the WT compared to the Zbtb16-/- BMDM treated with LPS and nigericin (Fig. 4d). Further evidence for ZBTB16-dependent assembly of the inflammasome was demonstrated by measuring the interaction between ASC and NLRP3 or Caspase-1 by immunoblotting of immunoprecipitants from the lysates of WT and Zbtb16-/- cells treated with nigericin (Fig. 4e).
