Phosphorylation of ERK1/2, JNK1/2, p38MAPK, I-κB, NLRP3 assembly and ASC oligomerization in macrophages. A, Protein levels of NLRP3, proIL-1β; (B) p-ERK1/2, p-JNK1/2, p-p38MAPK and (C) p-I-κB by Western blot analysis. Cells were incubated with Cf-02 at indicated concentrations for 30 minutes, followed by incubation with 1 μg/mL of LPS. NLRP3 assembly of (D) ASC-NLRP3; (E) NEK7-NLRP3; (F) PKR-NLRP3 by immunoprecipitation. G, ASC oligomerization by Western blot analysis. Cells were incubated with 1 μg/mL of LPS for 5.5 hours, and then with Cf-02 at concentrations indicated for 30 minutes, followed by 5 mM ATP for 30 minutes. Data are presented as means ± SEM with three separate experiments, and each experiment was performed in triplicate. Cf-02, a novel benzamide-linked small molecule; IB, immunoblotting; IP, immunoprecipitation