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. 2021 Oct 6;12:5862. doi: 10.1038/s41467-021-26142-w

Fig. 5. S803D phospho-mimetic mutation blocks NEK7 recruitment by NLRP3.

Fig. 5

a Endogenous NEK7 immunoprecipitates from LPS-primed BMDMs treated with nigericin (30 min) were analyzed for NLRP3 (Nlrp3+/++beads, Nlrp3+/+ BMDM lysate incubated with A/G-beads without anti-NEK7). b NLRP3-deficient U937 cells reconstituted with NLRP3 mutants were treated with PMA, doxycycline, LPS, and nigericin. Endogenous NEK7 immunoprecipitates were analyzed for NLRP3 (WT + IgG, lysate of U937 expressing WT NLRP3 incubated with isotype control and A/G-beads). c, d BMDMs were treated with LPS (6 h) and nigericin (30 min). Endogenous BRCC3 immunoprecipitates were analyzed for NLRP3 (Nlrp3+/++IgG, BMDM lysate incubated with IgG isotype control and A-beads; Brcc3−/−, BRCC3 immunoprecipitates from Brcc3−/− BMDM lysate) (c). NLRP3 ubiquitination was assessed by NLRP3 immunoprecipitation followed by anti-Ub WB (Nlrp3+/++beads, Nlrp3+/+ BMDM lysate incubated with A/G-beads without anti-NLRP3) (d). e NLRP3-deficient U937 cells reconstituted with NLRP3 mutants were treated with PMA and doxycycline (2 μg/ml, 16 h) followed by LPS (5 h) and nigericin (20 min). Endogenous BRCC3 immunoprecipitates were analyzed for NLRP3 (WT + IgG, lysate of U937 cells reconstituted with NLRP3 WT incubated with isotype control and A-beads). f NLRP3-deficient U937 cells reconstituted with NLRP3 mutants were treated with PMA and doxycycline (2 μg/ml, 16 h) followed by LPS (50 ng/ml, 4 h) and MG132 (10 μM, 40 min), E-64d (20 μg/ml, 40 min) and VX765 (2.5 μM, 40 min) 10 min before nigericin (30 min). NLRP3 ubiquitination was assessed by NLRP3 immunoprecipitation followed by anti-Ub WB. Caspase-1 inhibitor VX765 was added to prevent pyroptosis. g NLRP3 WT and S806D mutant were expressed with Myc-BRCC3 in 293T cells in the presence or not of HA-NEK7. Myc-BRCC3 immunoprecipitates were analyzed for NLRP3 (IgG, lysate of 293T cells expressing NLRP3 WT, Myc-BRCC3 and HA-NEK7 incubated with isotype control and A/G-beads). h Immortalized WT and Nek7−/− BMDMs were treated with LPS (6 h) and nigericin (30 min) in the presence of VX765 (2.5 μM, 15 min before nigericin). BRCC3 immunoprecipitates were analyzed for NLRP3 by WB (WT + IgG, lysates of immortalized WT BMDMs incubated with isotype control and A-beads). i, j BMDMs were treated with LPS (6 h) and nigericin (30 min) with oridonin (2 μM, 30 min before nigericin). Endogenous BRCC3 immunoprecipitates were analyzed for NLRP3 (i). NLRP3 ubiquitination was assessed by NLRP3 immunoprecipitation followed by anti-Ub WB (j). Data are one representative of three independent experiments. Molecular weights are indicated in kDa. Lys lysates, IP immunoprecipitates, Ub ubiquitin.