Rabbit anti sumo1

For detection of ubiquitinated and SUMOylated TOP3Accs,we performed DUST assay as described previously^{22 (link)}. Briefly, nucleic acids and covalent protein-nucleic acid adducts were recovered from FLAG-tagged R364W-TOP3A transfected cells using the RADAR assay. 10 μg of each RADAR assay sample was digested with 250 units benzonase nuclease (EMD Millipore, 100 units/μl) in the presence of 5 mM CaCl₂, followed by SDS-PAGE electrophoresis for immunodetection of total TOP3Accs and ubiquitinated and SUMOylated TOP3Accs by probing with rabbit anti-TOP3A (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP), mouse anti-ubiquitin antibody (dilution 1:500, Cell Signaling Technology, Cat# 3936), rabbit anti-SUMO-1 (dilution 1:1000, Cell Signaling Technology, Cat# 4940) and rabbit anti-SUMO-2/3 antibody (dilution 1:1000, Cell Signaling Technology, Cat# 4971), respectively.

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.