FIG 5.

ASFV pH240R inhibits the formation of NLRP3 inflammasome. (A) The effect of pH240R on the formation of NLRP3 inflammasome in HEK293T cells. HEK293T cells were transfected with a plasmid encoding Flag-NLRP3 alone or together with a plasmid expressing HA-ASC, HA-caspase-1, and HA-pH240R as indicated. At 24 hpt, co-IP was performed with anti-Flag beads. (B) The effect of pH240R on the formation of NLRP3 inflammasome in THP-1 cells. THP-1 or THP-1-pH240R cells were lysed, and then co-IP was performed with IgG or anti-NLRP3 antibody. (C) The relative band intensity analysis of the ASC/NLRP3 and caspase-1/NLRP3 immunoblotting result from panel B using ImageJ. (D) The effect of pH240R on the interaction of endogenous NLRP3 and ASC. CRL-2843 cells were transfected with empty vector or increasing amounts of a plasmid expressing Flag-pH240R and then treated with nigericin (5 μM). Then, 4 h later, the cells were collected to perform co-IP with IgG or anti-NLRP3 antibody. The immunoprecipitants and the whole-cell lysates were detected by Western blotting with the indicated antibodies. (E) The relative band intensity analysis of the ASC/NLRP3 immunoblotting result from panel D using ImageJ. All assays were independently repeated at least three times. The data presented are the mean ± SD. *, P < 0.05; ***, P < 0.001.