The interactions of endogenous ASC, ZBTB16, SUMO1 and NLRP3 were assessed in macrophage cells by proximity ligation assay (PLA, Duolink) according to the manufacturer’s instructions (Sigma-Aldrich). BMDMs were fixed with -20 °C methyl alcohol and then permeabilised with 0.3% Triton X-100 before the addition of the specified antibody pairs mouse anti-ASC (Santa Cruz) with rabbit anti-ZBTB16 (Bioss) or rabbit anti-SUMO1 (Cell Signalling Technology) or, alternatively, rabbit anti-ASC (AdipoGen) with mouse anti-NLRP3 (AdipoGen). The images were captured by Zeiss LSM 880 confocal fluorescence microscope at 63x magnification with Airyscan image processing and analyses with ImageJ software.
