FIG 7.

ASC ubiquitination at K110 and K140 is critical for porcine NLRP3 inflammasome-mediated IL-1β production. (A) HEK293T cells were transfected with plasmids expressing Myc-tagged NLRP3 and Flag-tagged WT/mutant ASC. At 24 hpt, the cells were fixed, permeabilized, immunostained with appropriate antibodies, and counterstained with DAPI. ASC specks formed by colocalization of NLRP3-Myc (gray) and Flag-ASC (green) were seen in the perinuclear region. Nuclei were visualized in blue. Scale bar, 5 μm. (B) HEK293T cells were transfected with plasmids expressing porcine NLRP3, Flag-tagged WT/mutant ASC, procaspase-1, and pro-IL-1β. At 12 hpt, the cells were infected with Hf09-817 at an MOI of 5 for 12 h. Porcine IL-1β was measured by ELISA with cell-free supernatants, and the expression of pro-IL-1β, p20, and viral proteins was measured by Western blotting with cell lysates. Expression of pro-IL-1β and p20 was normalized to that of β-actin using ImageJ. Results are representative of three independent experiments. **, P = 0.002.