(A) HEK293T cells were transfected with the Flag-NLRP3 and pcdna3.1(+)-ASC in combination without(-) or with (+) HA-3D. Lysates were either analyzed directly (30 μg protein, bottom) by immunoblotting using anti-Flag, anti-ASC, and anti-3D antibody (input) or subjected to IP using control IgG and anti-Flag antibody, and then analyzed by immunoblotting using anti-Flag, anti-ASC and anti-3D antibody (top). (B) HEK293T cells were transfected with pcdna3.1(+)-ASC, Flag-NLRP3, and HA-3D for 24 h, and then harvested and performed for ASC oligomerization assay. (C and D) HEK293T cells were transfected with Flag-NLRP3 plasmid in combination with plasmids encoding with GFP, GFP-3D, or HA-ASC. The localization of nucleus marker DAPI (blue), GFP or GFP3D (green), Flag-NLRP3 (red), HA-ASC (cyan), and merge were analyzed with confocal microscopy.