1. Volume 64, Issue 2, Pages 267-281 (October 2016)
USP38 Inhibits Type I Interferon Signaling by Editing TBK1 Ubiquitination through NLRP4 Signalosome 
Meng Lin, Zhiyao Zhao, Zhifen Yang, Qingcai Meng, Peng Tan, Weihong Xie, Yunfei Qin, Rong-Fu Wang, Jun Cui 
Molecular Cell 
Volume 64, Issue 2, Pages (October 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 64, 267-281DOI: (10.1016/j.molcel.2016.08.029)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
USP38 Negatively Regulates Type I IFN Signaling as well as Antiviral Responses
(A and B) Luciferase activity in 293T cells or 293T-TLR3 cells, transfected with a luciferase reporter for IFN-β (A) (IFN-β-luc) or for ISRE (B) (ISRE-luc), together with empty vector (EV) (no wedge) or an increasing dose of USP38, followed by treatment with or without intracellular (IC) low-molecular-weight poly(I:C) (pIC) (5 μg/mL) (IC pIC L), IC high-molecular-weight poly(I:C) (5 μg/mL) (IC pIC H), poly(dA:dT) (pdA:dT) (5 μg/mL), poly(I:C) (pIC) (10 μg/mL), or VSV-EGFP (MOI, 0.01) (VSV). Results are expressed relative to renilla luciferase activity.
(C) Luciferase activity in 293T cells transfected with control (Ctrl) siRNA or USP38-specific siRNAs, together with an ISRE-luc, then left untreated (UT) or treated with IC poly(I:C), poly(dA:dT), or VSV-eGFP is shown.
(D) Immunoblot analysis of total and phosphorylated (p-) IRF3 in THP-1 cells transfected with Ctrl siRNA or USP38-specific siRNA, followed by treatment with IC poly(I:C) or infection with VSV-EGFP at different time points is shown.
(E–G) Real-time PCR analysis of IFNα4 and IFNβ (E) and ELISA of IFN-β protein (F) and IFIT1, IFIT2, and CCL5 mRNA in THP-1 cells and human peripheral blood mononuclear cells (PBMCs) (G) treated with Ctrl siRNA or USP38-specific siRNAs, followed by infection with VSV-eGFP or IC poly(I:C) and analyzed at the indicated time points, are shown.
(H and I) Phase-contrast (PH) and fluorescence microscopy (H) and GFP intensity analyses (I) of A549 cells transfected with Ctrl siRNA or USP38-1 siRNA, and then infected with VSV-EGFP at the indicated MOI. Data in (A)–(C), (E)–(G), and (I) are expressed as means ± SEM of three independent experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, versus cells with the same treatment in WT cells, Student’s t test).
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
USP38 Deficiency Enhances Type I IFN Signaling as well as Antiviral Responses In Vivo
(A and B) Real-time PCR analysis of IFNβ (A) and ELISA of IFN- β protein (B) in BMMs from WT or Usp38−/− mice after VSV-EGFP or HSV-1 infection, at the indicated time points, is shown.
(C and D) Real-time PCR analysis of IFIT1, IFIT3, ISG15, and CXCL10 mRNA in BMMs from WT or Usp38−/− mice after VSV (C) or HSV-1 (D) infection is shown.
(E) Virus titers of WT or Usp38−/− BMMs after VSV or HSV-1 infection are shown.
(F) ELISA of cytokine production in sera from WT and Usp38−/− mice (n = 5 per group), which were intravenously injected with VSV, is shown.
(G) Determination of VSV loads in organs by TCID50 assay from WT and Usp38−/− mice is shown.
(H) Survival of 7-week-old WT and Usp38−/− mice given intravenously injection of VSV (1 × 108 PFU/g) (n = 6 per group; p < 0.01). Data in (A)–(H) are expressed as means ± SEM of three independent experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, versus cells with the same treatment in WT controls, Student’s t test).
See also Figure S2.
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5. Figure 3 USP38 Interacts with TBK1 after Viral Infection
(A) Luciferase activity of 293T cells transfected with an ISRE- or IFN-β-luc, together with RIG-I(N), MDA5, MAVS, TBK1, IKKi, or IRF3, along with EV (no wedge) or with increasing amounts (wedge) of USP38, is shown.
(B) Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with Myc-USP38 together with FLAG-tagged MAVS, TBK1, IKKi, IRF3, or IRF7 are shown.
(C) Confocal microscopy of USP38 and TBK1 in 293T cells transfected with GFP-USP38 and DsRed-TBK1 is shown. DAPI, DNA-intercalating dye.
(D–F) Co-immunoprecipitation and immunoblot analysis of 293T, THP-1, and PBMCs infected with VSV-EGFP for various times (above lanes) are shown.
(G) Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with HA-TBK1 and deletion mutants of FLAG-USP38 (FL, full-length; N, N-terminal without any conversed domain; C, C-terminal with UCH domain). Numbers above indicate amino acid position. Data from (A) are plotted as means ± SEM. Similar results were obtained in three independent experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, versus controls).
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6. Figure 4 USP38 Mediates the Degradation of Activated TBK1
(A) Immunoblot analysis (top) of extracts of 293T cells transfected with FLAG-TBK1 and HA-IRF3 and increasing doses of USP38 (wedge). RT-PCR analysis (bottom) of TBK1 mRNA is shown; GAPDH mRNA (encoding glyceraldehyde phosphate dehydrogenase) serves as a loading control.
(B) 293T cells were transfected with FLAG-tagged TBK1, IKKi, IKKα, IKKβ, and increasing doses of USP38 (wedge).
(C) Immunoassay of extracts of 293T cells transfected with FLAG-TBK1 and Myc-USP38 and treated with MG132, DMSO, or 3-Methyladenine (3-MA) is shown.
(D and E) Immunoblot analysis (D) and luciferase activity (E) of 293T cells transfected with FLAG-TBK1 and Myc-USP38, as well as USP38-specific or control siRNAs, together with an ISRE luciferase reporter, are shown.
(F and G) 293T cells were transfected with Myc-USP38 together with FLAG-TBK1 (WT), FLAG-TBK1 (S172A) or (S172E). After immunoprecipitation with anti-FLAG beads, Myc-USP38 was analyzed by immunoblot with anti-Myc (F). TBK1, TBK1-S172A, and their phosphorylation levels were determined by immunoblot (G). SA, S172A; SE, S172E.
(H) Immunoblot analysis of extracts of 293T cells transfected with EV or Myc-USP38, followed by infection with VSV-EGFP for the indicated time points, is shown.
(I) Immunoblot analysis of extracts of THP-1 cells transfected with Ctrl siRNA or USP38-specific siRNA, followed by infection with VSV-EGFP for the indicated time points with the indicated antibodies, is shown.
(J) Immunoblot analysis of extracts of mouse bone marrow macrophages (BMMs) from WT or Usp38−/− mice, after VSV or HSV-1 infection for the indicated time points with the indicated antibodies. Data in (E) are expressed as means ± SEM of three independent experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, versus cells with the same treatment in control cells, Student’s t test).
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
USP38 Mediates the K33-K48 Ubiquitination Transition of TBK1 on Lys 670
(A) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with various combinations of plasmid encoding FLAG-tagged TBK1, Myc-tagged USP38, and HA-tagged K48-linked or K63-linked ubiquitin, and treated with MG132, are shown.
(B) Immunoassay of extracts of 293T cells transfected with FLAG-tagged TBK1 and HA-tagged K48-linked ubiquitin, together with control siRNA or USP38-specific siRNA, assessed as in (A), is shown.
(C) Immunoblot analysis of 293T cells transfected with FLAG-TBK1 and Myc-USP38 (WT), Myc-USP38 (CA), Myc-USP38 (HA), or Myc-USP18 (CA/HA) is shown.
(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with various combinations of plasmid for FLAG-tagged TBK1, HA-tagged K33-linked ubiquitin, Myc-tagged USP38, or USP38 (CA/HA), and treated with MG132, are shown.
(E) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with various combinations of plasmid encoding Myc-tagged USP38, and HA-tagged K33-linked or HA-tagged K48-linked ubiquitin, then infected with VSV-EGFP for the indicated time points, are shown.
(F) Mass spectrometry analysis of a peptide derived from ubiquitinated TBK1 shows ubiquitin conjugation at K33 and K48 residues of ubiquitin.
(G) Ratio of K33 and K48 ubiquitin linkage in TBK1 immunoprecipitates, in WT and USP38−/− cells after VSV infection, is shown.
(H) Di-K33-linked ubiquitin (left) or HA-K33-linked ubiquitinated TBK1 (right) was incubated with immuno-purified FLAG-USP38 in vitro in deubiquitinating buffer. The immunoblot was probed with anti-HA.
(I) Confocal microscopy analysis of USP38 and K33-linked ubiquitin chains in 293T cells transfected with GFP-USP38 and DsRed-K33-linked ubiquitin by HSV-1 infection or UT is shown.
(J) Immunoblot analysis of extracts of 293T cells transfected with EV or Myc-USP38, together with FLAG-tagged WT TBK, K661R mutant, or K670R mutant of TBK1, is shown.
(K) Immunoprecipitation and immunoblot analysis of 293T cells transfected with FLAG-tagged WT TBK1 or K670R mutant of TBK1 and HA-tagged K33-linked or HA-tagged K48-linked ubiquitin are shown.
(L) Immunoprecipitation and immunoblot analysis of 293T cells transfected with various combinations (above lanes) of vectors encoding Myc-tagged USP38, HA-tagged K33-linked or K0-linked ubiquitin, and FLAG-tagged WT TBK1 or the K670R mutant of TBK1, and treated with MG132, are shown.
(M) Ratio of K33 and K48 ubiquitin linkage in WT FLAG-TBK1 and FLAG-TBK1(K670R) immunoprecipitates by mass spectrometry analysis.
See also Figure S4.
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8. Figure 6 USP38 Promotes TBK1 Degradation in an NLRP4-Dependent Manner
(A) Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with HA-USP38 together with FLAG-tagged NLRP4 or NLRP3 are shown.
(B and C) Immunoassay of extracts of THP-1 cells (B) and PBMCs (C) infected with VSV-EGFP for the indicated time points, followed by immunoprecipitation with anti-NLRP4 and immunoblot analysis with anti-USP38, is shown.
(D) Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with Myc-USP38 and deletion mutants of FLAG-NLRP4 are shown.
(E) Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with HA-NLRP4 and deletion mutants of FLAG-USP38 are shown.
(F) Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with HA-TBK1 and FLAG-USP38, as well as Ctrl or NLRP4-specific siRNA, are shown.
(G) Co-immunoprecipitation and immunoblot analysis of THP-1 cells infected with VSV-EGFP and harvested at the indicated time points are shown.
(H) Immunoblot analysis of 293T cells transfected with FLAG-TBK1 and Myc-USP38, as well as Ctrl or NLRP4-specific siRNA.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 NLRP4 Signalosome Regulates TBK1 Stability
(A and B) Immunoassay of extracts of 293T cells transfected with various combinations of plasmid encoding Myc-tagged TRIP and GFP-tagged DTX4, then infected with VSV-EGFP for the indicated time points, followed by immunoprecipitation with anti-TBK1 (A) or anti-NLRP4 (B) and immunoblotted with the indicated antibodies, is shown.
(C) Co-immunoprecipitation and immunoblot analysis of extracts of WT or USP38−/− 293T cells transfected with FLAG-TBK1, HA-NLRP4, HA-TRIP, and HA-DTX4 are shown.
(D) Co-immunoprecipitation and immunoblot analysis of extracts of WT or NLRP4−/− 293T cells transfected with FLAG-TBK1, HA-USP38, HA-TRIP and HA-DTX4 are shown.
(E and F) Immunoassays of extracts of WT or USP38−/− (E) or NLRP4−/− (F) 293T cells transfected with HA-tagged K33-linked or HA-tagged K48-linked ubiquitin, then infected with VSV-EGFP for the indicated time points, followed by immunoprecipitation with anti-TBK1 and immunoblot analysis with anti-HA, are shown.
(G and H) Immunoassays of extracts of WT or USP38−/− 293T cells transfected with EV, HA-NLRP4 (G), or HA-TRIP (H), followed by VSV-EGFP infection for the indicated time points, are shown.
(I) Immunoassay of extracts of USP38−/− 293T cells transfected with HA-NLRP4 together with EV, Myc-USP38, or Myc-USP38 (CA/HA), followed by IC poly(I:C) stimulation for 6 hr, is shown.
(J) Immunoassay of extracts of WT or NLRP4−/− 293T cells transfected with EV or HA-USP38, followed by VSV infection at the indicated time points.
See also Figures S6 and S7.
Molecular Cell  , DOI: ( /j.molcel )
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