Vangl2 inhibits NF-κB signaling by interacting with p65.
(A-C) Cho (A) or HEK293T cells (B and C) were co-transfected with a NF-κB and TK-Renilla reporter along with increasing amounts of Vangl2 for 18 h, then treated the cells with or without LPS (A, 250ng/ml), IL-1β (B, 40 ng/ml), or TNF-α (C, 20 ng/ml) for 6 h. NF-κB promoter driven luciferase activity was measured and normalized to the Renilla luciferase activity.
(D) Luciferase activity in HEK293T transfected with plasmids encoding an NF-κB luciferase reporter and TK-Renilla reporter, together with a vector encoding MyD88, IRAK1, TRAF6, IKKα, IKKβ, or p65, along with or without Vangl2 plasmid, was measured at 24 h after transfection and normalized to the Renilla luciferase activity.
(E) HEK293T cells were transfected with plasmids encoding HA-tagged Vangl2 and Flag-tagged p65, followed by immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA. Throughout was the immunoblot analysis of whole-cell lysates without immunoprecipitation.
(F) BMDMs were stimulated with LPS (100 ng/ml) for the indicated times. The cell lysates were subjected to immunoprecipitation with an anti-p65 antibody or control IgG, followed by immunoblotting with an anti-Vangl2 or anti-p65 antibody.
(G) The WT and Vangl2-deficient peritoneal macrophages were treated with LPS (1000 ng/ml) for 4 h, and co-localization of p65 and Vangl2 was detected by immunofluorescence (p65, green; Vangl2, red; DAPI, blue).
(H) A structural diagram of Vangl2 as well as schematic representation of Myc-tagged truncation mutants of Vangl2 (top). HEK293T cells were transfected with Flag-tagged p65 and empty vector, Myc-tagged Vangl2 (FL) or Vangl2 truncation mutants. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies (bottom).
(I) HEK293T cells were transfected with Flag-tagged p65 and HA-tagged Vangl2 FL or PkBD truncation. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(J) Luciferase activity in HEK293T cells transfected with an NF-κB luciferase reporter, together with a vector encoding p65, along with the empty vector or with vectors encoding Vangl2 or its truncation mutants. The results are presented relative to Renilla luciferase activity.
IP, immunoprecipitation; WCL, whole-cell lysate. Data are representative of three independent experiments and are plotted as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001 vs. corresponding control. NS, not significant.