USP1–UAF1 complex promotes stabilization of TBK1. (A) Western blot analysis of lysates from HEK293T cells transfected with V5-tagged ubiquitin (V5-Ub), HA-TBK1 and USP1 WT, USP1 C90S, or UAF1, followed by immunoprecipitation with anti-HA, probed with anti-V5. (B) Western blot analysis of lysates from HEK293T cells transfected with HA-tagged K48-linked ubiquitin (K48-Ub), Myc-TBK1 and USP1 WT, USP1 C90S, or UAF1, followed by immunoprecipitation with anti-Myc, probed with anti-HA. (C) Western blot analysis of lysates from HEK293T cells transfected with Myc-TBK1 and Flag-USP1, together with HA-K48-Ub or HA-K63-Ub, followed by immunoprecipitation with anti-Myc, probed with anti-HA. (D) Western blot analysis of lysates from mouse peritoneal macrophages treated with DMSO or 30 µM ML323 for 4 h and then infected with SeV for 8 h, followed by immunoprecipitation with anti-TBK1, and probed with anti-Ub. (E) Western blot analysis of extracts from HEK293T cells transfected with increasing amounts of USP1 or UAF1 expression plasmid. (F) Western blot analysis of extracts from mouse peritoneal macrophages treated with 30 µM ML323 for 4 h and then stimulated with LPS for the indicated time periods. (G and H) Western blot analysis of extracts from mouse peritoneal macrophages silenced of USP1 or UAF1 and then stimulated for various times with SeV (G) or LPS (H). (I and J) Immunoblot analysis of extracts from DMSO or 30 µM ML323 pretreated mouse peritoneal macrophages stimulated with LPS for 4 h and then treated for various times with CHX. TBK1 and IRF3 expression levels were quantitated by measuring band intensities by using ImageJ software. The values were normalized to actin (J). Similar results were obtained in three independent experiments.