IFI16 protein binds to AIM2 protein in the cytoplasmic fraction and increased levels of the IFI16 protein inhibits the AIM2-ASC inflammasome-mediated activation of casapase-1.

<p>(<b>A</b>) Cultures of human embryonic kidney cells (HEK-293) at ∼80% confluency were either transfected with pCMV-AIM2 plasmid (2 µg) allowing the expression of the FLAG-tagged human AIM2 protein or along with the pCMV-IFI16 plasmid (1 µg) allowing the expression of IFI16 protein. 40 h after the transfections, cells were fractionated into cytoplasmic and nuclear fractions and fractions containing approximately equal amounts of proteins were subjected to immunoprecipitation using specific antibodies to the FLAG-tag. The immunoprecipitates were analyzed by immunoblotting using antibodies to the FLAG-tag (the upper panel) or the IFI16 protein (the lower panel). The fold change (FC) in the IFI16 protein levels in immunoprecipitates is also indicated. (<b>B</b>) <u>Left panel:</u> HEK-293 cells were either left untransfected (lane 1) or transfected with plasmid DNAs encoding for the ASC (DNA 50 ng; lanes 2–5), CASP-1 (DNA 100 ng; lanes 2–5), AIM2 (200 ng; lanes 3–5), or IFI16 (200 or 400 ng; lanes 4 and 5 respectively) proteins. Total amount of plasmid DNA in the transfections was adjusted to 2 µg using the pCDNA3 plasmid DNA. About 40 h after the transfections, cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins. <u>Right panel:</u> Relative caspase-1 (p20) protein levels shown in the left panel (lanes 1–4).