CARD8 fails to bind CAPS-associated NLRP3 mutants. (A) Diagrammatic representation of the three-domain structure of NLRP3 with asterisks indicating the location of mutations. (B) Human Embryonic Kidney (HEK)293 cells were transfected with expression plasmids (500 ng each) for NLRP3-Flag (wild type (WT), R260W, D303N or H312P) and caspase recruitment domain-containing protein 8 (CARD8) with or without apoptosis-associated speck-like protein containing a CARD (ASC) (300 ng). At 24 hours after transfection, expression of each protein (Input) was confirmed by Western blotting. Simultaneously, the whole cell lysates were the immunoprecipitated (IP) with anti-Flag antibody and analyzed by Western blotting. (C) Similar experiments as above were carried out, but CARD8 was Flag-tagged in place of NLRP3. (D) HEK293 cells were transfected with expression plasmids of proIL-1β (200 ng), NLRP3 (WT, R260W, D303N, H312P or N477K) (50 ng), procaspase-1 (10 ng), ASC (25 ng) and CARD8 or an empty vector (100 ng). At 24 hours after transfection, the supernatants were replaced by fresh medium and incubated for another 24 hours; thereafter, the second supernatants were subjected to ELISA and pellets were analyzed by Western blotting. Levels of IL-1β secretion from transfectants were expressed as a relative percentage compared with those from cells with each NLRP3 without CARD8. Each column represents the mean ± SD (n = 3), ***P <0.001 vs. no CARD8. NS, not significant vs. no CARD8.