1. Volume 21, Issue 6, Pages 1613-1623 (November 2017)
The E3 Ubiquitin Ligase TRIM40 Attenuates Antiviral Immune Responses by Targeting MDA5 and RIG-I 
Chunyuan Zhao, Mutian Jia, Hui Song, Zhongxia Yu, Wenwen Wang, Qi Li, Lining Zhang, Wei Zhao, Xuetao Cao 
Cell Reports 
Volume 21, Issue 6, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 1613-1623DOI: (10.1016/j.celrep.2017.10.020)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 TRIM40 Negatively Regulates RLR-Induced IFN-β Expression
(A) ELISA analysis of IFN-β, TNF-α, and IL-6 expression in peritoneal macrophages from Trim40+/+ or Trim40–/– mice after infected with SeV or VSV for 12 hr or stimulated with LPS or poly(I:C) for 4 hr. All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(B) qPCR analysis of IFN-β mRNA expression in Trim40+/+ MEFs, Trim40–/– MEFs, or Trim40–/– MEFs transfected with TRIM40 plasmid, following infection with SeV for 12 hr. All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(C) ELISA analysis of IFN-β expression in MEFs from Trim40+/+ or Trim40–/– mice after transfected with poly(I:C) for 4 hr. All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(D) HEK293T cells were transiently transfected with IRF3, NF-κB, or ISRE reporter plasmid, together with an increasing amount of TRIM40 expression plasmid or control plasmid, and then infected with SeV for 12 hr. All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(E) western blot analysis of the indicated phosphorylated and total signaling proteins in peritoneal macrophages from Trim40+/+ or Trim40−/− mice infected with SeV.
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4. Figure 2 TRIM40 Deficiency Enhances Antiviral Immune Responses
(A and B) Trim40+/+ MEFs, Trim40–/– MEFs, or Trim40–/– MEFs transfected with TRIM40 (WT) or TRIM40 ΔRING were infected with VSV-GFP (1 MOI) for 12 hr, and then they were imaged by microscopy (A) or analyzed for VSV replication by qPCR (B). Scale bar, 100 μm.
(C and D) Trim40+/+ or Trim40−/− mice were intraperitoneally injected with VSV (4 × 107 PFU) for 12 hr (n = 5 per group). ELISA of cytokine production in serum (C) and qPCR analysis of VSV replication in liver and lung (D) are shown. All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(E) Trim40+/+ or Trim40−/− mice (n = 4 per group) were intraperitoneally injected with VSV (4 × 107 PFU) for 48 hr. H&E staining of lung tissue sections is shown. Scale bar, 50 μm.
(F) Survival of Trim40+/+ and Trim40−/− mice (n = 10 per group) intraperitoneally infected with VSV (1 × 108 PFU). All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(G) ELISA of cytokine production in serum of Trim40+/+ or Trim40−/− mice that were intraperitoneally injected with EMCV (100 PFU) for 12 hr (n = 6 per group). All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(H) Survival of Trim40+/+ and Trim40−/− mice (n = 12 per group) intraperitoneally infected with EMCV (1 × 104 PFU). All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
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5. Figure 3
TRIM40 Interacts with CARD of MDA5 and RIG-I through Its CC Domain
(A) HEK293T cells were transiently transfected with IFN-β reporter plasmid and adaptor plasmids as indicated, together with TRIM40 expression plasmid or control plasmid, and analyzed for luciferase activity. All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(B) Lysates from HEK293T cells transiently transfected with FLAG-TRIM40 and Myc-MDA5, RIG-I, or MAVS were subjected to immunoprecipitation with anti-FLAG antibody followed by western blot analysis with anti-Myc antibody. Proteins in whole-cell lysate were used as a positive control (Input).
(C and D) Lysates from mouse peritoneal macrophages infected with SeV for the indicated time periods were subjected to immunoprecipitation with anti-MDA5 (C) or anti-RIG-I (D) antibody, followed by western blot analysis with the indicated antibody.
(E) Schematic diagram of TRIM40 and its truncate mutants. R, RING domain; B, B box; CC, coiled-coil region.
(F) FLAG-tagged TRIM40 or its mutants and Myc-MDA5 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody and then immunoblotted with the indicated antibodies.
(G) Schematic diagram of MDA5 and its truncate mutants. C1, CARD1; C2, CARD2; ATP, ATP-binding domain; CTD, C-terminal domain.
(H) Myc-tagged MDA5 or its mutants and FLAG-TRIM40 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody and then immunoblotted with the indicated antibodies.
(I) Schematic diagram of RIG-I and its truncate mutants. N218 is also termed as RIG-I CARD.
(J) HA-tagged RIG-I mutants and FLAG-TRIM40 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-HA antibody and then immunoblotted with the indicated antibodies.
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6. Figure 4 TRIM40 Promotes Proteasomal Degradation of MDA5 and RIG-I
(A) western blot analysis of the indicated signaling proteins in peritoneal macrophages from Trim40+/+ or Trim40−/− mice infected with SeV for the indicated time periods.
(B and C) western blot analysis of lysates from Trim40+/+ or Trim40−/− mouse peritoneal macrophages infected with SeV for 12 hr and then treated for various times with cycloheximide (CHX) (B). MDA5 and RIG-I expression levels were quantitated by measuring band intensities using ImageJ software (C). All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(D) western blot analysis of lysates from HEK293T cells transfected with MDA5 plasmid and an increasing amount of TRIM40 plasmid (0, 0.5, 1, or 2 μg) or TRIM40 ΔRING.
(E) western blot analysis of lysates from HEK293T cells transfected with RIG-I plasmid, along with an increasing amount of TRIM40 plasmid (0, 0.5, 1, or 2 μg), and then treated with MG132 (10 μM) for 4 hr.
(F) western blot analysis of lysates from HEK293T cells transfected with MDA5 plasmid and TRIM40 plasmids (WT or ΔRING) and then treated with MG132 (10 μM) or chloroquine (100 μM) for 4 hr.
Cell Reports  , DOI: ( /j.celrep )
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7. Figure 5
TRIM40 Promotes K27- and K48-Linked Ubiquitination of MDA5 and RIG-I
(A) Lysates from HEK293T cells transiently cotransfected with HA-Ub, FLAG-TRIM40, along with Myc-MDA5 were subjected to immunoprecipitation with anti-Myc antibody followed by western blot analysis with anti-HA antibody.
(B and C) Lysates from HEK293T cells transiently cotransfected with HA-Ub, FLAG-TRIM40 (WT and C29S), and Myc-MDA5 (B) or Myc-RIG-I (C) were immunoprecipitated using anti-Myc antibody. The immunoprecipitates were denatured and reimmunoprecipitated with anti-Myc antibody (two-step immunoprecipitation [Re-IP]), and then they were analyzed by western blot.
(D and E) TRIM40, TRIM40 C29S, MDA5, and RIG-I were obtained by in vitro transcription and translation. In vitro ubiquitination assay was performed in the presence of Ub, E1, UbCH5a, TRIM40, and MDA5 (D) or RIG-I (E). The ubiquitination of MDA5 or RIG-I was examined by western blot with anti-Myc antibody.
(F) Re-IP analysis lystes from HEK293T cells transiently cotransfected with HA-Ub (WT and its mutants), FLAG-TRIM40, and Myc-MDA5.
(G and H) MEFs from Trim40+/+ or Trim40−/− mice were infected with SeV for 8 hr. Cell lysates were immunoprecipitated with anti-MDA5 (G) or anti-RIG-I (H) antibody, followed by western blot analysis with the indicated antibodies.
Cell Reports  , DOI: ( /j.celrep )
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8. Figure 6
CARD Is Required for TRIM40-Mediated Inhibition of Type I IFN Signaling
(A) HEK293T cells were transiently transfected with IFN-β reporter plasmid, MDA5 CARD (MDA5 mutant containing only CARD), or RIG-I CARD (RIG-I mutant containing only CARD), together with TRIM40 (WT or ΔRING mutant) expression plasmids or control plasmid, and analyzed for luciferase activity. All data are represented as means ± SD (∗p < 0.05 and ∗∗p < 0.01).
(B and C) western blot analysis of lysates from HEK293T cells transfected with MDA5 CARD (B) or RIG-I CARD (C) plasmid, along with an increasing amount of TRIM40 plasmid (0, 0.5, 1, or 2 μg), and then treated with DMSO or MG132 (10 μM) for 4 hr.
(D and E) Lysates from HEK293T cells transiently cotransfected with Ub, FLAG-TRIM40, along with MDA5 CARD (D) or RIG-I CARD (E) were subjected to immunoprecipitation with the indicated antibody, followed by western blot analysis.
(F and G) Lysates from HEK293T cells transiently cotransfected with HA-Ub, FLAG-TRIM40, along with Myc-MDA5 (WT and its point mutants) were subjected to Re-IP with anti-Myc antibody, followed by western blot analysis.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions