Fig 2: TRIM18 recruits PPM1A to dephosphorylate TBK1 for its inactivation and black the interactions of TBK1 with its upstream adaptors. a Immunoblot (IB) analysis of total and phosphorylated (p-) IRF3 as well as β-actin in lysates of WT and TRIM18 KO BMDM infected for 1 h without (Mock) or with CVB3 or adenovirus at an MOI of 5. b Immunoblot analysis of endogenous proteins TRIM18 and PPM1A precipitated with anti-PPM1A, or immunoglobulin G (IgG) from whole-cell lysates of WT and TRIM18 KO BMDM. c Schematic diagram showing full-length PPM1A (Full) and serial truncations of PPM1A with deletion of various domain (left margin); numbers at ends indicate amino acid positions (top). M1, the catalytic domain; M2, the C-terminal no catalytic domain. d Immunoblot analysis of purified HA-tagged full-length PPM1A and serial truncations of PPM1A with deletion of various domains alone with anti-HA antibody (top blot) or after incubation with Myc-tagged TRIM18 and immunoprecipitation with anti-Myc antibody (second blot), and immunoblot analysis of purified Myc-tagged TRIM18 with anti-Myc antibody (third blot) or after incubation with Myc-tagged TRIM18 and immunoprecipitation with anti-Myc antibody (bottom blot). e Schematic diagram showing full-length TRIM18 (Full) and serial truncations of TRIM18 with deletion (Δ) of various domain (left margin); numbers at ends indicate amino acid positions (top). RING, the really interesting new gene domain; BBOX, the B-box zinc-finger domain; BBC, the B-box C-terminal domain; FN3, the fibronectin type 3 domain; SPRY, the Sp1A kinase and Ryanodine receptors domain. f Immunoblot analysis of purified HA-tagged full-length TRIM18 and serial truncations of TRIM18 with deletion of various domains alone with anti-HA antibody (top blot) or after incubation with Myc-tagged PPM1A and immunoprecipitation with anti-Myc antibody (second blot), and immunoblot analysis of purified Myc-tagged PPM1A with anti-Myc antibody (third blot) or after incubation with Myc-tagged PPM1A and immunoprecipitation with anti-Myc antibody (bottom blot). g Immunoblot analysis of total and phosphorylated (p-) TBK1, PPM1A, TRIM18 as well as β-actin in lysates of WT and TRIM18 KO BMDM infected for 1 h without (Mock) or with CVB3 or adenovirus at an MOI of 5. h Immunoblot analysis of endogenous proteins STING, MAVS, TBK1 and TRIM18 from whole-cell lysates (Input) or precipitated with anti-TBK1 antibody (IP: TBK1) from lysates of WT and TRIM18 KO BMDM left infected (Mock) or infected with CVB3 or adenovirus at MOI of 10 for 6 h. The position of protein markers (shown in kDa) is indicated on the right. Data are representative of three independent experiments

Fig 3: TRIM18 inhibits type I IFN production by human THP-1 macrophages in response to stimulations with dsRNA and dsDNA or infections with RNA and DNA viruses. a The immunoblot (IB) analysis of TRIM18, MAVS or STING expression in human THP-1 macrophages treated with shRNA to knockdown expression of TRIM18, MAVS or STING. A scrambled shRNA served as a control (sh-Ctrl) and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the loading control. The position of protein markers (shown in kDa) is indicated at right. b-h, ELISA of IFN-β production from human THP-1 macrophages treated with the indicated shRNA after a 12 h stimulations with dsRNA including 5’pppRNA (0.5 μg/ml) b and poly I:C (0.5 μg/ml) c or dsDNA from HSV-1 virus (HSV60, 2.5 μg/ml) d delivered by Lipofectamine 3000, or 12 h infections with RNA viruses including influenza A virus (influenza A virus PR8 strain, Flu PR8) e and Coxsackievirus B3 (CVB3) (f), or DNA viruses including HSV-1 g and adenovirus h at an MOI of 2 (n = 3 per group). Each circle represents an individual independent experiment and small solid black lines indicate the average of triplicates for results in (b–h). Mock, scrambled shRNA-treated cells without stimulation. NS, not significant (p > 0.05), **p < 0.01, ***p < 0.001, and p value was calculated by unpaired two-tailed Student’s t test. Data are representative of three independent experiments