FIG 6.

K110 and K140 in the C terminus of porcine ASC are targeted for ubiquitination. (A) A schematic representation of WT and two mutant ASC constructs (aa 1 to 109 and aa 1 to 158) is illustrated with lysine (K) residues. (B to D) HEK293T cells were transfected with HA-ubiquitin and Flag-tagged WT or mutant ASC constructs. At 24 hpt, the cells were infected with Hf09 WT or Hf09-817 at an MOI of 10 for 9 h. Cell lysates under denaturing conditions were subjected to co-IP with beads conjugated with anti-Flag antibody. IP and input samples were used for Western blotting with antibodies indicated. (E) HEK293T cells were transfected with HA-tagged WT or mutant ubiquitin and Flag-ASC constructs for 24 h and infected as described for panel B. Co-IP and Western blotting were done as described for panel B. The asterisks indicate IgG heavy chains. Results are representative of three independent experiments.