Fig. 1

IKKβ binds NLRP3 to induce rapid inflammasome assembly and pyroptotic cell death. a–d HEK293 cells were transiently cotransfected with HA-tagged full-length IKKβ, Flag-tagged full-length or mutant NLRP3, and Flag-tagged full-length NLRC4, AIM2, or NLRP1. Shown are representative immunoblots depicting the binding of IKKβ to NLRP3, but not NLRC4, AIM2, and NLRP1 (a, b), and specific domains of NLRP3 (c, d). n = 3 to 4 independent experiments. e Determination of colocalization between IKKβ and NLRP3 in BMDMs by confocal microscopy. Shown are representative immunostainings of three independent experiments. Scale bar = 20 µm. f Representative immunoblot showing the assessment of NLRP3 oligomerization in transfected HEK293 cells stimulated with nigericin and analyzed by blue native PAGE. n = 3 independent experiments. g THP-1 macrophages were either stimulated with LPS (200 ng/mL) for 4 h and nigericin (5 µM) for 60 min or ATP (5 mM) for 30 min or left unstimulated. Shown is a representative immunoblot depicting the endogenous binding of IKKβ to NLRP3. n = 3 independent experiments. h Protein–protein interactions between IKKβ and NLRP3 were analyzed in solution applying microscale thermophoresis (MST). 50 nM MST-Red-NLRP3 was titrated against increasing concentrations of IKKβ. Plotted is the fraction of bound MST-Red-NLRP3 (fraction bound) over the indicated concentrations of IKKβ (log scale). Data are represented as mean ± SD, with 3–4 data points for each concentration of IKKβ. Two independent titrations measured in two separate sets of capillaries. i–q Simultaneous engagement of TLRs and NLRP3. BMDMs were treated with TPCA-1 (500 nM) for 1 h and were simultaneously stimulated with LPS (200 ng/mL) and nigericin (5 µM) for 1 h. i Experimental outline. j Representative immunoblot of NLRP3 Oligomerization. n = 4 independent experiments. k Representative immunoblot depicting caspase-1 cleavage. l Quantification of cleaved caspase-1 normalized to actin. n = 5 independent experiments. m Representative immunoblots of GSDMD cleavage. n Quantification of GSDMD cleavage normalized to actin. n = 6 independent experiments. o Measurement of LDH release. n = 3 independent experiments. p Determination of Zombie uptake. Shown are representative immunostainings of three independent experiments. Scale bar = 50 µm. q Quantification of Zombie uptake. Data are represented as mean ± SEM.Two-sided unpaired t test was used in the statistical analyses after testing for normality with Shapiro–Wilk-Test