Fig 2: UBXN3B regulates STING dimerization, phosphorylation, and degradation. a Immunoblots showing Sting dimerization in untreated (Ubxn3b+/+) and 4-hydroxyl tamoxifen-induced Cre+/− Ubxn3bflox/flox (Ubxn3b−/−) primary MEFs. The cells were infected without (mock) or with HSV-1 (MOI = 0.5) for 8 h. b Immunoblotting analysis of the whole-cell lysates of bone marrow-derived cDCs infected with HSV-1 (MOI = 5). Mono monomer, 2-ME β-mercaptoethanol, a chemical compound that reduces disulfide bonds. c qPCR quantification of Ifnb1 and Tnfa mRNA induction in cDCs by HSV-1 (MOI = 5). Bars: mean ± s.e.m. Two biological replicates were pooled for qPCR (N = 2 per genotype per time point). *P < 0.05 (unpaired Student’s t test). d–f Immunoblotting analysis of the whole-cell lysates of d MEFs infected with HSV-1 (MOI = 0.5), e MEFs transfected with ISD (8 µg/ml) in the absence or presence of 40 µM of chloroquine (+CQ) and f trophoblasts transfected with cGAMP (8 µg/ml). In e, f, the arrows indicate phosphorylated STING with long and short exposure. Actin, Tubulin (Tub), and GAPDH are housekeeping protein controls. g qPCR quantification of IFNB1 and TNFA mRNA induction in trophoblasts transfected with cGAMP (8 µg/ml). In c, g, the bars are: mean ± s.e.m. Two biological replicates were pooled for qPCR (N = 2 per genotype per time point). *P < 0.05 (unpaired Student’s t test). The results are representative of two to three independent experiments

Fig 3: UBXN3B interacts with STING and TRIM56. a Co-immunoprecipitation (co-IP) of STING with UBXN3B from HEK293 cells transfected with FLAG-UBXNs and HA-STING plasmids using anti-FLAG magnetic beads, followed by immunoblotting (IB) with an anti-HA (STING) and FLAG (UBXN) antibody. b Immunofluorescence staining of STING and UBXN3B in H1975 cells treated without (Mock) or with ISD (8 µg/ml) for 3 h. The cells were stained with a mouse anti-human STING and rabbit anti-UBXN3B antibody followed by a secondary antibody conjugated with Alexa Fluor 594/488. The nuclei were stained with DAPI. Objective, ×40. Scale bar, 200 µm. c A schematic diagram of UBXN3B functional domains. d Co-IP of STING with the truncated forms of UBXN3B. The procedures are similar to b. e Co-IP of UBXN3B with TRIM from HEK293 cells transfected with FLAG-UBXN3B and Myc-TRIM plasmids using anti-Myc magnetic beads, followed by IB with an anti-Myc (TRIM) and FLAG (UBXN3B) antibody. f Co-IP of TRIM56 with the truncated forms of UBXN3B from HEK293 cells transfected with FLAG-UBXN3B truncates and Myc-TRIM56 plasmids using anti-FLAG magnetic beads, followed by IB with an anti-Myc (TRIM56) and FLAG (UBXN truncates) antibody. GAPDH is a housekeeping protein control. WCE whole-cell extract. The results are representative of two independent experiments

Fig 4: UBXN3B is critical for STING-dependent IFN-I induction in mouse primary cells. a ELISA of IFN-α in the cell culture supernatants of (Ubxn3b+/+, Ubxn3b−/−) bone marrow-derived macrophages (Mϕ), Flt3-induced pDCs, and GM-CSF-induced cDCs (pooled from five littermates) 20 h after the indicated treatments. N = 3 per genotype. *P < 0.05 (unpaired Student’s t test). b Immunoblots of an interferon-stimulated gene Oas1a, Sting, and Ubxn3b expression in cDCs 20 h after the indicated treatments. Tubulin is a housekeeping protein control. qPCR analysis of (c) Ifnb1 and Tnfa mRNA expression and d cellular HSV-1 genome loads in cDCs infected with HSV-1 (MOI = 10) for the indicated time. e Immunoblots showing Ubxn3b and housekeeping Gapdh protein expression in mock (Ubxn3b+/+) or 4-hydroxyl tamoxifen-treated Cre+/− Ubxn3bflox/flox (Ubxn3b−/−) MEFs. f Viral titers (plaque-forming units/ml) in the supernatant of MEFs infected with HSV-1 (MOI = 0.1). N = 3 per genotype per time point. *P < 0.05; **P < 0.01 (unpaired Student’s t test). g qPCR analysis of selected immune gene mRNA expression in MEFs infected with HSV-1 as in g. h The immunoblots show knockout efficacy of STING and UBXN3B by CRISPR-Cas9 in human primary trophoblasts. Actin is a housekeeping protein control. i Fluorescent microscopic images of human primary trophoblasts infected with HSV-1-GFP (MOI = 0.3) for 18 h. Objective, ×5. Scale bar, 10 µm. j qPCR analysis of Ifnb1 mRNA expression in human primary trophoblasts infected with HSV-1-GFP for the indicated time. Bars/data points: mean ± s.e.m. Two biological replicates were pooled for qPCR (N = 2 per genotype per time point). *P < 0.05; **P < 0.01 (unpaired Student’s t test). The results are representative of two independent experiments

Fig 5: UBXN3B is critical for type I IFN responses to SeV and VSV, but not EMCV infection. a The survival curves of Ubx3b−/− (N = 6) and Ubxn3b−/− (N = 7) mice infected with 1 × 107 PFU of VSV i.v. The results are pooled from two experiments. *P < 0.05 (log-rank test). b, c qPCR analysis of Ifnb1 expression in cDCs infected with b VSV (MOI = 5) or c SeV (200 hemagglutination units per 105 cells). d ELISA of IFN-α in the cell culture supernatants of cDCs infected with SeV and VSV as in b, c. N = 3 per genotype per time point. *P < 0.05 (unpaired Student’s t test). e The survival curves of Ubx3b+/+ (N = 7) and Ubxn3b−/− (N = 8) mice infected with 200 PFU of EMCV i.p. P = 0.53 (log-rank test). The results are pooled from two experiments. f qPCR analysis of Ifnb1 expression in cDCs infected with EMCV (MOI = 5) for the indicated time. Bars/data points: mean ± s.e.m. Two biological replicates were pooled for qPCR (N = 2 per genotype per time point). *P < 0.05 (unpaired Student’s t test). The results are representative of two independent experiments