FIGURE 4.

149-01 irreversibly, directly, and specifically binds to NLRP3. (A) IL-1β releases in supernatants from LPS-primed BMDMs treated with indicated doses of 149-01 for 15 min, washed three times before nigericin stimulation were detected by ELISA. (B) LPS-primed BMDMs cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot using antibodies for NLRP3, NEK7, pro-caspase-1, ASC and β-actin. (C) Binding affinity of 149-01 to purified GFP-NLRP3 protein was analyzed by MST. HEK-293T cells transfected with Flag-tagged NLRP3 (D), NLRP1b (E), AIM2 (F), or NLRC4 (G) were lysed and the cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m (n = 6) (A), or are representative of three independent experiments (B–G). One-way ANOVA was applied to calculate statistical significance: ***p < .001.