Interaction of CARD8 with full length NLRP3. (A) Diagrammatic representation of the dual domain structure of caspase recruitment domain-containing protein 8 (CARD8). (B) Human Embryonic Kidney (HEK)293 cells were cotransfected with 500 ng each of expression plasmid for NLRP3 and either CARD8-Flag, function-to-find (FIIND)-Flag, CARD-Flag or an empty vector. At 24 hours after transfection, whole cell lysates were immunoprecipitated (IP) with anti-Flag antibody and analyzed by Western blotting. The total amount of the plasmid DNA in all transfection reactions was kept constant by adding the empty vector plasmid. (C) HEK293 cells were transfected with expression plasmids for NLRP3-Flag (500 ng), CARD8 (500 ng) and apoptosis-associated speck-like protein containing a CARD (ASC) (10, 30, 100 or 300 ng), and analyzed as above.