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. 2020 Aug 9;12(4):261–278. doi: 10.1007/s13238-020-00751-5

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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TBK1 phosphorylates HDAC3 to promote its deacetylase activity. (A) Immunoblot analysis of phosphorylated HDAC3 by TBK1 in HEK293T cells (1.5 × 10⁶) transiently co-transfected for 48 h with Flag-tagged HDAC3 and empty vectors or HA-tagged TBK1 expression plasmids before co-immunoprecipitation (with anti-Flag or IgG as a control) and immunoblot analysis (with antibody to anti-Phospho-HDAC3 (Ser424), anti-Phospho-Threonine, anti-HA or anti-Flag). (B) In vitro kinase assay of phosphorylated HDAC3. The peptide of HDAC3 full length protein as the substrate and combinations of BSA or recombinant protein TBK1 were introduced as indicated and the immunoblot analysis with antibody to anti-Phospho-HDAC3 (Ser424), anti-Phospho-Threonine, anti-HDAC3 or anti-TBK1. (C) Immunoblot analysis of HEK293T cells (2 × 10⁵) transiently co-transfected for 48 h with HA-tagged TBK1 (1 μg) and Flag-tagged HDAC3 (300 ng), followed by infection with or without SeV for 6 h. (D) HDAC3 was phosphorylated by TBK1. HEK293T cells (1.5 × 10⁶) were transiently co-transfected for 48 h with Flag-tagged HDAC3 and empty vectors or HA-tagged TBK1 expression plasmids, and treated with GSK8612 for 1 h before co-immunoprecipitation (with anti-Flag or IgG as a control) and the immunoblot analysis with antibody to anti-Phospho-Threonine, anti-HA, anti-Flag or anti-phospho-TBK1. (E) Analysis of HDAC3 enzymatic activity in the presence of recombinant TBK1 protein or TSA (negative control). (F) Immunoblot analysis of phosphorylated and total HDAC3, TBK1 or IRF3 and β-actin of HEK293T cells infected with SeV for 0–12 h (left) and densitometric analysis (right). Data are representative of three independent experiments. Graphs show mean ± SD; n = 3. **P < 0.01 (Student’s t-test)