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. Author manuscript; available in PMC: 2023 Apr 15.
Published in final edited form as: J Immunol. 2022 Mar 30;208(8):2029–2036. doi: 10.4049/jimmunol.2101154

FIGURE 3.

FIGURE 3.

The Q132R mutation confers NEK6 the ability of NLRP3 binding and inflammasome activation. (A) HA-tagged mouse wild-type (WT), mutant NEK6 Q132R, or Y136H was expressed alone or with Flag-tagged mouse NLRP3 in HEK 293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies. (B) NEK7 KO iBMDMs were reconstituted with HA-tagged mouse wild-type (WT) or Q132R mutant NEK6 by lentiviral transduction. Macrophages were stimulated with 5 μM nigericin for 1 h after LPS priming. Mixtures of cell lysates and supernatants were immunoblotted with indicated antibodies. EV, empty vector. Actin was used as an internal control. (C) Measurement of IL-β in the supernatants from nigericin-stimulated macrophages by ELISA. (D) ASC immunostaining in wild-type (WT) or Q132R mutant NEK6-reconstituted macrophages after nigericin stimulation. Scale bars, 10 μm. Data are representative of three experiments. Error bars in C show the SEM of triplicate wells. *p < 0.05.