Figure 3. UAF1 and USP1 interact with... | Rockefeller University Press

Figure 3.

$Figure 3. UAF1 and USP1 interact with TBK1. (A) HEK293T cells were transfected with RIG-I, MDA5, MAVS, TRIF, TBK1, and IRF3 5D along with UAF1 or USP1 plasmid and IFN-β reporter plasmid, and luciferase activity was analyzed. (B) HEK293T cells were transfected with TBK1 along with UAF1 or USP1 plasmid, and IRF3, ISRE, or NF-κB reporter plasmid, and luciferase activity was analyzed. (C) Western blot analysis of UAF1 and USP1 expression in mouse peritoneal macrophages stimulated with LPS for the indicated time periods. (D) Western blot analysis of USP1 expression in nuclear or cytoplasmic fractions of mouse peritoneal macrophages stimulated with LPS for the indicated time periods. (E) Lysates from HEK293T cells transiently cotransfected with Flag-USP1 and Myc-TBK1, Myc-IRF3, Myc-TRAF3, or Myc-RIP1 expression plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot analysis with anti-Myc antibody. (F) Lysates from HEK293T cells transiently cotransfected with Flag-UAF1 and Myc-IRF3, Myc-TBK1, Myc-RIP1, or Myc-TRAF3 expression plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot analysis with anti-Myc antibody. (G) Lysates from HEK293T cells transiently cotransfected with Myc-TBK1 and Flag-UAF1 or Flag-USP1 expression plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot analysis with anti-Myc antibody. (H–J) Lysates from mouse peritoneal macrophages stimulated with LPS were subjected to immunoprecipitation with the indicated antibody or control IgG followed by Western blot analysis with the indicated antibodies. Proteins in whole-cell lysate were used as positive control (Input). Data are representative of three experiments (mean ± SD of six samples in A and B; **, P < 0.01; by Student’s t test.). Similar results were obtained in three independent experiments in C–J.$

UAF1 and USP1 interact with TBK1. (A) HEK293T cells were transfected with RIG-I, MDA5, MAVS, TRIF, TBK1, and IRF3 5D along with UAF1 or USP1 plasmid and IFN-β reporter plasmid, and luciferase activity was analyzed. (B) HEK293T cells were transfected with TBK1 along with UAF1 or USP1 plasmid, and IRF3, ISRE, or NF-κB reporter plasmid, and luciferase activity was analyzed. (C) Western blot analysis of UAF1 and USP1 expression in mouse peritoneal macrophages stimulated with LPS for the indicated time periods. (D) Western blot analysis of USP1 expression in nuclear or cytoplasmic fractions of mouse peritoneal macrophages stimulated with LPS for the indicated time periods. (E) Lysates from HEK293T cells transiently cotransfected with Flag-USP1 and Myc-TBK1, Myc-IRF3, Myc-TRAF3, or Myc-RIP1 expression plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot analysis with anti-Myc antibody. (F) Lysates from HEK293T cells transiently cotransfected with Flag-UAF1 and Myc-IRF3, Myc-TBK1, Myc-RIP1, or Myc-TRAF3 expression plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot analysis with anti-Myc antibody. (G) Lysates from HEK293T cells transiently cotransfected with Myc-TBK1 and Flag-UAF1 or Flag-USP1 expression plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot analysis with anti-Myc antibody. (H–J) Lysates from mouse peritoneal macrophages stimulated with LPS were subjected to immunoprecipitation with the indicated antibody or control IgG followed by Western blot analysis with the indicated antibodies. Proteins in whole-cell lysate were used as positive control (Input). Data are representative of three experiments (mean ± SD of six samples in A and B; **, P < 0.01; by Student’s t test.). Similar results were obtained in three independent experiments in C–J.

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