Extended Data Fig. 7: c-di-AMP binds NLRP3.

(a) Biotin or biotin-labelled c-di-AMP were incubated with BMDM cell lysates for 4 h, followed by precipitation using streptavidin beads. Precipitates were immunoblotted with antibodies against NLRP3 and two known NLRP3 binding partners. (b) Wild-type BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cells were lysed and immunoprecipitated with a control IgG or antibody against NLRP3. Precipitates were subjected to an ELISA assay for determining the c-di-AMP levels. (c) BMDM cells from Nlrp3^+/+ and Nlrp3^–/– mice were primed with 1 μg/ml LPS for 3 h, followed by transfection of 10 μg/ml c-di-AMP using DOTAP for 6 h. Cells were lysed and pellets were crosslinked with 2 mM disuccinimidyl suberate (DSS) for 30 min at room temperature. Crosslinked pellets were dissolved in 1 X Laemmli buffer and immunoblotted with antibody against ASC. Cell lysates were also immunoblotted as indicated. (d) Biotin-labelled c-di-AMP were incubated with recombinant NLRP3 protein for 2 h and crosslinked by ultraviolet, followed by precipitation using antibody against NLRP3. Precipitates were immunoblotted with antibody against biotin. (e) Wild-type BMDM cells were transfected with saline or 10 μg/ml c-di-AMP using DOTAP for 6 h, followed by immunostaining with antibody against NLRP3. Nuclei were stained with DAPI. (f) Plasmids encoding NLRP3 and ADCY7 without transmembrane domains were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against NLRP3. Precipitates were immunoblotted as indicated. (g) Plasmids encoding FLAG-tagged NLRP3 PYD domain (aa 1–134) and ADCY7 without transmembrane domains were co-transfected into HEK293T cells for 24 h, followed by immunoprecipitation with a control IgG or antibody against FLAG. Precipitates were immunoblotted as indicated. In e, scale bar, 10 μm. In b, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.

Source data