Fig. 3. NLRP3 K24 acetylation promotes the inflammasome assembly.

a–c Co-IP analysis of interaction between NLRP3 with ASC, NEK7, or NLRP3 from HEK293T cells transfected with myc-tagged ASC, NEK7, or NLRP3 together with Flag-tagged WT NLRP3 or its nonacetylation mimetic (K-to-R) mutations. d Immunoblot analysis of NLRP3 oligomerization and expression by SDD-AGE and SDS-PAGE assay in HEK293T cells transfected with Flag-tagged WT NLRP3 or its nonacetylation mimetic (K-to-R) mutations plasmid and then stimulated with nigericin (10 μM, 1 h). e, f COS-7 cells transfected with Vector-GFP, NLRP3 (WT or K24R)-GFP plasmids and stimulated with nigericin (10 μM, 2 h). e Representative fluorescent microscopy living-cell images of NLRP3 oligomerization. Scale bar = 20 μm. f Ratio of different size of GFP puncta in GFP⁺ cells. n = 3 biologically independent experiments. For videos of three representative cells, see Supplementary movies 1, 2 and 3. g–i SDD-AGE, BN-PAGE and 2D-SDS-PAGE analysis of NLRP3 oligomers of BMDMs from Nlrp3^WT/WT or Nlrp3^K24/K24R mice treated with LPS (100 ng/mL, 3 h) along or with ATP (5 mM, 1 h) or nigericin (10 μM, 1 h). Results are represented as mean ± SD and typical photographs are representative of three biological independent experiments with similar results. Statistical analyses were carried out via two-way ANOVA with the Bonferroni test for (f). Source data are provided as a Source Data file.