Figure 7: IRF8 interacts physically with RORγ.

(a) 293T cells were transfected with HA-tagged IRF8 and T7-tagged RORγt overexpression plasmids for 40 h and cell lysates were prepared in the presence or absence of DNase I or ethidium bromide. 500 μg of cell lysates were immunoprecipitated with an anti-T7 antibody and immunoblotted with specific antibodies as indicated. Data are representative of three independent experiments. (b) NIH3T3 cells were transiently transfected with GFP-IRF8 and T7-RORγt for 40 h, and cells were fixed and stained red for RORγt followed by confocal microscopic analysis. Scale bar, 50 μm. (c) Naive CD4⁺ T cells from WT mice were cultured under T_H17-polarizing conditions for 72 h and the expression of RORγt and IRF8 was analysed by flow cytometry. The cells were gated on CD4⁺ T cells. Data are representative of three independent experiments. (d) Naive CD4⁺ T cells from wild-type or Irf8^–/– mice were cultured under T_H17-polarizing conditions for 60 h and the cell lysates were then immunoprecipitated with an anti-IRF8 antibody and western blotted (WB) with anti-RORγt and anti-IRF8 antibodies. Data represent three independent experiments. (e) Diagrams of IRF8 protein domains. (f) 293T cells were co-transfected with plasmids containing Flag-tagged full-length IRF8, IRF8 fragments (1–230, 1–190, 1–154) and T7-tagged RORγt plasmid for 40 h, and co-immunoprecipitation using anti-Flag antibody from the cell extracts was performed and immunoblotted with anti-T7 antibody. Data are representative of three independent experiments. (g) 293T cells were co-transfected with an IL-17 promoter reporter construct containing the 6-kbp promoter, a RORγt plasmid and either a full-length IRF8 or the IRF8 truncation mutant (1–114) construct for 30 h. Luciferase assays were performed as described in b. Data indicate mean±s.d. of triplicate cultures and are representative of three independent experiments. *P<0.05 versus cells transfected with IRF8 mutant (Student's t-test).