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. Author manuscript; available in PMC: 2021 Jul 11.
Published in final edited form as: Nat Microbiol. 2021 Jan 11;6(3):401–412. doi: 10.1038/s41564-020-00832-5

Figure 4. Pak1 phosphorylates NLRP3 to promote inflammasome activation.

Figure 4

(a) In vitro [γ-32P]-ATP kinase assay using human recombinant NLRP3 (arrows) and human recombinant Pak1 (arrowheads) analyzed by autoradiography and Coomassie Brilliant Blue (CBB) staining. (b) HEK293T cells were transfected with plasmids encoding NLRP3 inflammasome component (ASC-GFP, mCaspase-1), pro-IL-1β-Flag, myc-Pak1T423E, with either myc-NLRP3 or myc-NLRP3 S163A or myc-NLRP3 S198A or myc-NLRP3 T659A or myc-NLRP3 S163A, S198A, T659A and IL-1β maturation was analyzed by immunoblot. (c) HEK293T cells were transfected with plasmids encoding NLRP3 inflammasome components (ASC-GFP, mCaspase-1), myc-Pak1T423E, with either myc-NLRP3 or myc-NLRP3 T659A or myc-NLRP3 T659D and IL-1β maturation was analyzed by immunoblot. (d-e) NLRP3 knock-out iBMDMs reconstituted either with NLRP3 or NLRP3 T659A were treated with vehicle or LPS (100 ng/mL) and CNF1 (500ng/mL) for 8h, (d) supernatants and cell lysates were analyzed by immunoblot, (e) supernatants were analyzed by ELISA for IL-1β (n=4 biologically independent samples) and TNF-α (n=3 biologically independent samples). Data are expressed as the mean ± SEM. Statistical analyses were performed using a two-tailed unpaired Student’s t-test. (f) HEK293T cells were transfected with plasmids encoding either myc-NLRP3 or myc-NLRP3 S163A or myc-NLRP3 S198A or myc-NLRP3 T659A or myc-NLRP3 S163A, S198A, T659A. Cell lysates were processed for anti-myc immunoprecipitation and endogenous Nek7 was revealed using an anti-Nek7 antibody. (g) NLRP3 knock-out iBMDMs reconstituted either with NLRP3 or NLRP3 T659A were treated with CNF1 (500 ng/mL) for 6 h. Cell lysates were processed for anti-Nek7 or isotypic IgG immunoprecipitation. The numbers on the side of the immunoblots indicate molecular weight (kDa). Experiments were repeated at least three times, and representative data are shown.