(a) Endogenous NEK7-NLRP3 association in LPS-primed J774A.1 cells treated with the indicated inhibitors, stimulated with nigericin, and analyzed by immunoprecipitation and immunoblot. (b,c) Endogenous NEK7-NLRP3 association was analyzed by immunoprecipitation and immunoblot. The phosphorylation state of NEK7 was analyzed using Phos-tag SDS-PAGE. (b) J774A.1 cells primed with LPS and stimulated with ATP were lysed and treated with calf intestinal alkaline phosphatase (CIP). (c) J774A.1 cells primed with LPS and stimulated with ATP together with N-acetylcysteine (NAC). (d) ELISA analysis of IL-1β in the culture supernatants of J774A.1 cells treated as in c. The means of triplicate samples are plotted. Results are representative of two independent experiments.