Fig. 4. NLRP11^PYD recruits ASC and is necessary for efficient ASC polymerization.

a,b, Immunoprecipitation with immobilized anti-Flag (a) and anti-HA (b) antibodies, and TCLs were analyzed by immunoblot for HA-, Myc- and Flag-tagged proteins after transient transfection of HEK293 cells with Flag-ASC^PYD, HA-NLRP11^PYD and HA-NLRP3^PYD (a) and HA-NLRP11^PYD, Myc-ASC^PYD and Myc-NLRP3^PYD (b), as indicated. c, Immunoprecipitation with immobilized anti-ASC antibodies from TCL of NLRP11^KO cells restored with NLRP11^Flag or NLRP11^ΔPYD-Flag left untreated, primed with Pam3CSK4 (1 μg ml⁻¹, 2 h) and primed and activated with nigericin (5 μM, 10 min); immunoprecipitates and TCLs were analyzed by immunoblot for Flag, ASC and tubulin loading control. Arrowheads indicate the correct-size protein. d, IL-1β, IL-18 and TNF ELISA from SNs of NLRP11^KO cells restored with low-expressing NLRP11^Flag cells (NLRP11^lo, NLRP11^ΔPYD(lo)), high-expressing NLRP11^ΔPYD(hi) and ASC^KD cells left untreated, primed with Pam3CSK4 (1 μg ml⁻¹, 4 h) and primed and activated with nigericin (5 μM, 25 min) (n = 3, mean ± s.d.); *P < 0.0001, **P = 0.0002, ***P = 0.0007, ****P = 0.0038. The dotted line indicates that for ASC^KD, only the Pam3CSK4 + nigericin group is shown. e, Fluorescence microscopy of EGFP and DAPI in HEK293^ASC-EGFP cells transiently transfected with empty vector (Ctrl), NLRP3 and NLRP11 as indicated (left) and quantification of ASC speck⁺ cells per view (right). (n = 5, mean ± s.d.); *P < 0.0001, scale bars, 100 μm. f, immunoblot for ASC of crosslinked TCL from above cells. Arrowheads indicate oligomers. g,h, Immunoblot for ASC of TCLs and crosslinked TCLs from Cas9^Ctrl, NLRP11^KO#1, NLRP11^KO#2 and NLRP3^KO cells left untreated or primed with LPS (200 ng ml⁻¹, 4 h) and activated with nigericin (5 μM, 20 min) (g) and NLRP11^Flag, NLRP11^ΔPYD-Flag and ASC^KD cells left untreated or primed with Pam3CSK4 (1 μg ml⁻¹, 4 h) and activated with nigericin (5 μM, 15 min) (h). Arrowheads indicate oligomers.

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