Figure 5. Cardiolipin binds to Nlrp3.

(A, B) J774A.1 cells were radiolabeled for 24 hrs with ¹⁴C linoleic acid (10 μCi); cells were then LPS-primed and stimulated for 12 hrs with ATP or silica. The mitochondrial fraction was isolated, solubilized in 1% Triton X-100 containing lysis buffer and subjected to immunoprecipitation with anti-Nlrp3 antibody or isotype control antibody. Lipids associated with the immunoprecipitates were extracted with chloroform: methanol (2:1 v/v). Radioactivity associated with the organic fraction following immunoprecipitation was quantified (A) and subjected to 1-D HPTLC; the location of migration of cardiolipin (CL) was determined by running a cardiolipin standard in parallel (B). (A) Results are expressed as the mean ± SEM of three independent experiments; ** p < 0.01, *** p < 0.001 by two-tailed unpaired Student's t-test. (C, D) LPS-primed BMM were treated with staurosporine, silica or nigericin for 8 hrs. Cell lysates and supernatants were collected and lysates assessed for caspase-1 and caspase-3 via immunoblot (C) and supernatants for IL-1β via ELISA (D); determinations were performed in triplicate and expressed as the mean ± SD. (E) Lipid strips were incubated with lysates of RAW264.7 cells expressing HA-tagged mouse Nlrp3 or control buffer; strips were then washed and developed with a mouse anti-HA antibody followed by anti-mouse IgG-HRP. (F) Lipid strips were incubated with 10 μg/ml of His-tagged human Nlrp3, Nlrc4 or TLR4; strips were then washed and developed with a mouse anti-His antibody followed by anti-mouse IgG-HRP. (G) The indicated phospholipid-coated beads were incubated with 10 μg/ml of His-tagged Nlrp3; following washes, bound and unbound His tagged-Nlrp3 was determined by immunoblotting with an anti-Nlrp3 antibody. (H) Lipid strips were incubated with lysates of HEK293T cells expressing Flag-tagged full length Nlrp3.1-1036, or truncation mutants Nlrp3ΔLRR.1-719, PYD.1-95, NACHT.69-546 or LRR.535-719; strips were then washed and developed with a mouse anti-FLAG antibody followed by anti-mouse IgG-HRP. Results are representative of two (C, E, F, H), three (B, G) or four (D) separate experiments. See also Figure S3.