Figure 3.

Deficiency of HDAC3 impairs phosphorylation of IRF3 by TBK1. (A) Luciferase activity (upper) in shNC or shHDAC3 cell lines (1 × 10⁵), transfected with an IFN-β-Luc, together with control vector (VEC), HA-tagged RIG-I, VISA, TBK1, IRF3, or IRF3-5D; the protein expression of RIG-I, VISA, TBK1, IRF3, or IRF3-5D were confirmed by immunoblotting analysis (lower). (B–D) Immunoblot analysis of phosphorylated and total TBK1 or IRF3 in shHDAC3 HEK293T cell lines (B), THP-1 HDAC3 deficient cell lines (C), HDAC3^fl/fl or HDAC3^fl/fl Lyz2-Cre BMDMs (D), followed by infection with SeV for the indicated times. (E) Immunoblot analysis of IRF3 in dimerization in HDAC3-deficient THP-1 cells and left infected with SeV for the indicated times, followed by native PAGE. (F) The interaction between HDAC3 and TBK1. HEK293T cells (1.5 × 10⁶) were co-transfected with the indicated plasmids (3 μg each) for 48 h. Co-immunoprecipitation and immunoblotting were performed with the indicated antibodies. (G) Endogenous HDAC3 dynamically interacts with TBK1, followed by infection with SeV. BMDMs were left uninfected or infected with SeV for the indicated times. Co-immunoprecipitation and immunoblotting were performed with the indicated antibodies. Data are representative of three independent experiments. Graphs show mean ± SD; n = 3. ns, no significant differences;*P < 0.05; ***P < 0.001 (Student’s t-test)