Figure 2.
M013 mutants interact with the differential affinity with ASC-1 and NF-κB1. A and B, expression (input) of V5-tagged WT-M013 and M013 mutants in the THP-1 cells transfected and infected with vMyx-M013KO and harvested 6 h (A) and 24 h (B) postinfection. C and D, detection of WT-M013 and its mutant's interaction with endogenous ASC-1 in THP1 cells by co-immunoprecipitation. After transfection and infection, co-IP was performed using anti-V5 antibody, and the interacting endogenous ASC-1 in the complex was detected using anti-ASC-1 antibody by Western blotting analysis. The samples were prepared 6 h (C) and 24 h (D) after infection. E and F, detection of WT-M013 and its mutant's interaction with endogenous NF-κB1 (p105/p50) in THP1 cells by co-immunoprecipitation. After transfection and infection, co-IP was performed using anti-NF-κB1 antibody, and the interacting expressed V5-tagged M013 proteins in the complex were detected using anti-V5 antibody by Western blotting analysis. The samples were prepared 6 h (E) and 24 h (F) post infection. Lane M, protein marker; lane WT, WT M013; C1, cells infected with vMyx-M013KO; lane M013PYD, M013 with C terminus deletion; lanes E28Q, D29N, D57N, and D41A, M013 with mutations E28Q, D29N, D57N, and D41A, respectively; lane C2, cell lysates with no transfection or infection; *, a lane was deleted in the figure because of loading sample not described in the manuscript.