FIG 5.

NS1 C terminus of pandemic H1N1 inhibits ASC ubiquitination. (A and B) HEK293T cells were transfected with HA-vector or HA-ubiquitin (Ub) and Flag-vector or Flag-NLRP3/ASC constructs. At 24 hpt, the cells were infected with WT or NS1-mutant Hf09 virus 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. (C) HEK293T cells were transfected with HA-vector or HA-ubiquitin and Flag-ASC constructs along with plasmids expressing GFP, Myc-tagged WT, or mutant NS1 of Hf09. At 24 hpt, the cells were infected with Hf09-817 at an MOI of 10 for 9 h. Co-IP was done as described for panel A, and Western blotting was done with the antibodies indicated. (D) HEK293T cells were transfected with HA-ubiquitin and Flag-tagged human ASC (Flag-huASC) constructs for 24 h and infected with WT Hf09 or Hf09-817 at an MOI of 10 for 9 h. Co-IP was done as described for panel A, and Western blotting was done with the antibodies indicated. The asterisks indicate IgG heavy chains. Results are representative of three independent experiments.