Cross-linking and Imaging of ASC Speck Formation

WT and Nox4^−/− BMDMs (5 × 10⁶ cells in 100 mm cell culture dish) were harvested and lysed in 500 µl of lysis buffer (20 mM HEPES–KOH, pH 7.5, 150 mM KCl, 1% NP-40) as described previously^{29 (link),30 (link)}. Lysates were centrifuged at 330 × g for 10 min at 4 °C. The pellets were washed in 1 ml of PBS and resuspended in 500 µl of PBS. 2 mM disuccinimydyl suberate (DSS) was added to the resuspended pellets, which were incubated for 30 min with rotation at room temperature. Samples were then centrifuged at 330 × g for 10 min at 4 °C. The supernatant was removed, and the cross-linked pellets were resuspended in 50 µl of Laemmli sample buffer. Samples were analyzed by immunoblot analysis using polyclonal ASC antibody (ADI-905-173-100, Enzo Lifesciences). WT and Nox4^−/−BMDMs were seeded on chamber slides. After stimulation, cells were fixed with 4% paraformaldehyde and then incubated with polyclonal ASC antibody (ADI-905-173-100, Enzo Lifesciences) for 16 h followed by DAPI (P36962, ThermoFisher Scientific) staining as described previously^{29 (link),30 (link)}. ASC specks were analyzed by Zeiss LSM880 laser scanning confocal microscope and quantified using ImageJ software. The graph represents the quantification of cells with specks in five distinct areas.