Suppression of NLRP3 inflammasome by expression of CARD8. (A) Human Embryonic Kidney (HEK)293 cells were transfected with expression plasmids for proIL-1β (200 ng), NLRP3 (50 ng), procaspse-1 (10 ng), apoptosis-associated speck-like protein containing a CARD (ASC) (25 ng) and either caspase recruitment domain-containing protein 8 (CARD8)-Flag, function-to-find (FIIND)-Flag, CARD-Flag or empty vector (100 ng each). 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. Each column represents the mean ± SD (n = 4), **P <0.005, and ***P <0.001 vs. no CARD8 component. (B) HEK293 cells were transfected with the same set of plasmids as A. A total of 24 hours after transfection, or after a further two-hour incubation in serum-free medium, cell lysates (Lys) or supernatants (Sup), respectively, were analyzed by Western blotting.