p tbk1  (Abcam)

 
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    Name:
    Anti NAK TBK1 phospho S172 antibody EPR2867 2
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    ab109272
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    Structured Review

    Abcam p tbk1
    Mediation of the maturation and activation of BMDCs during Mycobacterium bovis infection by the cGAS pathway. Mycobacterium bovis regulated the maturation and activation of BMDCs via the <t>cGAS/STING/TBK1/IRF3-dependent</t> pathway and promoted type I IFN production. Furthermore, type I IFN and its receptor IFNAR contributed to this process. Moreover, the mature and activated BMDCs enhanced the proliferation of T cells by some surface markers and cytokines. These signaling pathways linked the innate and adaptive immune responses. (The full line arrows signify promotion; the dotted arrows signify releasing cytokines.)

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    Images

    1) Product Images from "cGAS/STING/TBK1/IRF3 Signaling Pathway Activates BMDCs Maturation Following Mycobacterium bovis Infection"

    Article Title: cGAS/STING/TBK1/IRF3 Signaling Pathway Activates BMDCs Maturation Following Mycobacterium bovis Infection

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20040895

    Mediation of the maturation and activation of BMDCs during Mycobacterium bovis infection by the cGAS pathway. Mycobacterium bovis regulated the maturation and activation of BMDCs via the cGAS/STING/TBK1/IRF3-dependent pathway and promoted type I IFN production. Furthermore, type I IFN and its receptor IFNAR contributed to this process. Moreover, the mature and activated BMDCs enhanced the proliferation of T cells by some surface markers and cytokines. These signaling pathways linked the innate and adaptive immune responses. (The full line arrows signify promotion; the dotted arrows signify releasing cytokines.)
    Figure Legend Snippet: Mediation of the maturation and activation of BMDCs during Mycobacterium bovis infection by the cGAS pathway. Mycobacterium bovis regulated the maturation and activation of BMDCs via the cGAS/STING/TBK1/IRF3-dependent pathway and promoted type I IFN production. Furthermore, type I IFN and its receptor IFNAR contributed to this process. Moreover, the mature and activated BMDCs enhanced the proliferation of T cells by some surface markers and cytokines. These signaling pathways linked the innate and adaptive immune responses. (The full line arrows signify promotion; the dotted arrows signify releasing cytokines.)

    Techniques Used: Activation Assay, Infection

    The cyclic GMP-AMP synthase (cGAS) pathway is activated in bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis infection. ( A ) BMDCs were treated with siRNA, siCon, and M. bovis , and cGAS protein was analyzed by Western blotting at 24 h after treatment. ( B ) The related proteins in the cGAS pathway in BMDCs were assayed by Western blotting. The protein levels of cGAS, p-STING, STING, TBK1, and p-TBK1 were analyzed in BMDCs transfected with siCon or sicGAS and then infected for 24 or 48 h with M. bovis (MOI 5). ( C ) The co-localization of IRF3 within the nucleus was detected by immunofluorescence microscopy (400 ×). ( D ) The culture supernatants were harvested after 24 h and assessed by ELISA. All data are expressed as mean ± SD, (* p
    Figure Legend Snippet: The cyclic GMP-AMP synthase (cGAS) pathway is activated in bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis infection. ( A ) BMDCs were treated with siRNA, siCon, and M. bovis , and cGAS protein was analyzed by Western blotting at 24 h after treatment. ( B ) The related proteins in the cGAS pathway in BMDCs were assayed by Western blotting. The protein levels of cGAS, p-STING, STING, TBK1, and p-TBK1 were analyzed in BMDCs transfected with siCon or sicGAS and then infected for 24 or 48 h with M. bovis (MOI 5). ( C ) The co-localization of IRF3 within the nucleus was detected by immunofluorescence microscopy (400 ×). ( D ) The culture supernatants were harvested after 24 h and assessed by ELISA. All data are expressed as mean ± SD, (* p

    Techniques Used: Derivative Assay, Infection, Western Blot, Transfection, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay

    2) Product Images from "A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity"

    Article Title: A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity

    Journal: Journal of Virology

    doi: 10.1128/JVI.00149-18

    The EALQR motif influences SeV-mediated activation of the RIG-I signaling pathway. (A to C) The EALQR deletion in the NS1 ED weakened the inhibition of SeV-induced IFN-β, ISRE, and NF-κB promoter activities. The protocol for the luciferase reporter assay is described in Materials and Methods. The lower immunoblots show the expression levels of the transfected NS1 or NS1Δ protein. WB, Western blot. (D) The EALQR deletion in the NS1 ED impaired the inhibition of SeV-induced transcription of antiviral genes. The qPCR protocol is described in Materials and Methods. (E) The EALQR deletion in the NS1 ED decreased the inhibition of SeV-induced phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were transfected with a control, NS1, or NS1Δ plasmid. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (F) The EALQR deletion in the NS1 ED decreased the inhibition of SeV-induced phosphorylation and dimerization of IRF3. HEK293 cells were transfected with a control, NS1, or NS1Δ plasmid. Twenty-four hours after transfection, the cells were mock infected or infected with SeV for 12 h. Cell lysates were separated by native PAGE (top) or SDS-PAGE (bottom) and analyzed by immunoblotting with the indicated antibodies. (G) Effects of NS1 and NS1Δ on VSV-GFP replication. HEK293 cells were transfected with the NS1 or NS1Δ plasmid. Twenty-four hours later, the cells were infected with VSV-GFP (MOI = 0.1). The cells were viewed microscopically, and the immunoblots were analyzed with the indicated antibodies.
    Figure Legend Snippet: The EALQR motif influences SeV-mediated activation of the RIG-I signaling pathway. (A to C) The EALQR deletion in the NS1 ED weakened the inhibition of SeV-induced IFN-β, ISRE, and NF-κB promoter activities. The protocol for the luciferase reporter assay is described in Materials and Methods. The lower immunoblots show the expression levels of the transfected NS1 or NS1Δ protein. WB, Western blot. (D) The EALQR deletion in the NS1 ED impaired the inhibition of SeV-induced transcription of antiviral genes. The qPCR protocol is described in Materials and Methods. (E) The EALQR deletion in the NS1 ED decreased the inhibition of SeV-induced phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were transfected with a control, NS1, or NS1Δ plasmid. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (F) The EALQR deletion in the NS1 ED decreased the inhibition of SeV-induced phosphorylation and dimerization of IRF3. HEK293 cells were transfected with a control, NS1, or NS1Δ plasmid. Twenty-four hours after transfection, the cells were mock infected or infected with SeV for 12 h. Cell lysates were separated by native PAGE (top) or SDS-PAGE (bottom) and analyzed by immunoblotting with the indicated antibodies. (G) Effects of NS1 and NS1Δ on VSV-GFP replication. HEK293 cells were transfected with the NS1 or NS1Δ plasmid. Twenty-four hours later, the cells were infected with VSV-GFP (MOI = 0.1). The cells were viewed microscopically, and the immunoblots were analyzed with the indicated antibodies.

    Techniques Used: Activation Assay, Inhibition, Luciferase, Reporter Assay, Western Blot, Expressing, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Infection, SDS Page, Clear Native PAGE

    ELQR-4A mutations attenuate inhibition of SeV-mediated activation of the RIG-I signaling pathway. (A) The ELQR-4A mutations weakened the inhibition of SeV-induced transcription of antiviral genes. HEK293 cells were transfected with a control, NS1, or NS1-4A expression plasmid. Twenty-four hours after transfection, the cells were infected with SeV or left uninfected for 12 h before quantitative PCR was performed. (B) The ELQR-4A mutations attenuated the blockade of SeV-induced phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were transfected with a control, NS1, or NS1-4A plasmid. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (C) The ELQR-4A mutations did not inhibit RIG-I ubiquitination. HEK293 cells were transfected with the indicated plasmids for 24 h and then subjected to immunoprecipitation with anti-HA antibodies; immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-HA antibody. Whole-cell lysates were analyzed by immunoblotting with anti-Flag antibodies. (D and E) The EALQR motif is required for NS1 multimerization. HEK293 cells were cotransfected with Flag- and HA-tagged NS1 and NS1 mutants. Twenty-four hours after transfection, the cells were mock infected or infected with SeV for 18 h, and then coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies.
    Figure Legend Snippet: ELQR-4A mutations attenuate inhibition of SeV-mediated activation of the RIG-I signaling pathway. (A) The ELQR-4A mutations weakened the inhibition of SeV-induced transcription of antiviral genes. HEK293 cells were transfected with a control, NS1, or NS1-4A expression plasmid. Twenty-four hours after transfection, the cells were infected with SeV or left uninfected for 12 h before quantitative PCR was performed. (B) The ELQR-4A mutations attenuated the blockade of SeV-induced phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were transfected with a control, NS1, or NS1-4A plasmid. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (C) The ELQR-4A mutations did not inhibit RIG-I ubiquitination. HEK293 cells were transfected with the indicated plasmids for 24 h and then subjected to immunoprecipitation with anti-HA antibodies; immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-HA antibody. Whole-cell lysates were analyzed by immunoblotting with anti-Flag antibodies. (D and E) The EALQR motif is required for NS1 multimerization. HEK293 cells were cotransfected with Flag- and HA-tagged NS1 and NS1 mutants. Twenty-four hours after transfection, the cells were mock infected or infected with SeV for 18 h, and then coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies.

    Techniques Used: Inhibition, Activation Assay, Transfection, Expressing, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, SDS Page, Immunoprecipitation

    An EALQR motif in the NS1 effector domain is a key determinant for type I IFN inhibition. (A) UV-inactivated supernatants from cells infected with rescued influenza virus were added to fresh confluent A549 cells for 24 h. The cells were then infected with VSV-GFP (MOI = 0.01). GFP expression was examined under a fluorescence microscope at 12 and 24 h postinfection. (B) Schematic diagram of recombinant LaSota strains. The NS1 and NS1Δ genes were inserted between the P and M genes of the LaSota genome by using a unique PmeI restriction enzyme site. (C and D) Replication of rescued recombinant LaSota viruses. rLaSota, rLaSota-NS1, or rLaSota-NS1Δ was inoculated into 7-day-old embryonic eggs for the indicated times or used to infect A549 cells for 24 h. Viral titers were calculated as EID 50 per milliliter, or the cells were stained and analyzed with a LaSota-specific antibody. (E) Effects of two NS1 proteins on the phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were infected with rLaSota, rLaSota-NS1, or rLaSota-NS1Δ (MOI = 1) for the indicated times before immunoblot analysis.
    Figure Legend Snippet: An EALQR motif in the NS1 effector domain is a key determinant for type I IFN inhibition. (A) UV-inactivated supernatants from cells infected with rescued influenza virus were added to fresh confluent A549 cells for 24 h. The cells were then infected with VSV-GFP (MOI = 0.01). GFP expression was examined under a fluorescence microscope at 12 and 24 h postinfection. (B) Schematic diagram of recombinant LaSota strains. The NS1 and NS1Δ genes were inserted between the P and M genes of the LaSota genome by using a unique PmeI restriction enzyme site. (C and D) Replication of rescued recombinant LaSota viruses. rLaSota, rLaSota-NS1, or rLaSota-NS1Δ was inoculated into 7-day-old embryonic eggs for the indicated times or used to infect A549 cells for 24 h. Viral titers were calculated as EID 50 per milliliter, or the cells were stained and analyzed with a LaSota-specific antibody. (E) Effects of two NS1 proteins on the phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were infected with rLaSota, rLaSota-NS1, or rLaSota-NS1Δ (MOI = 1) for the indicated times before immunoblot analysis.

    Techniques Used: Inhibition, Infection, Expressing, Fluorescence, Microscopy, Recombinant, Staining

    3) Product Images from "ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response"

    Article Title: ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008457

    Identification of ZFYVE1 as a negative regulator of MDA5-triggered signaling. (A) Knockdown of ZFYVE1 enhances transcription of downstream antiviral genes induced by EMCV but not SeV or VSV. HFFs or THP1 cells (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi plasmids were left un-infected or infected with EMCV, VSV or SeV for the indicated times (HFFs) or 6 h (THP1) before qPCR analysis of mRNA levels of the indicated genes. (B) Knockdown of ZFYVE1 enhances transcription of downstream antiviral genes induced by transfection of poly(I:C)-HMW but not poly(I:C)-LMW. HFFs (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi plasmids were mock- transfected or transfected with poly(I:C)-HMW (2 μg/ml) or poly(I:C)-LMW (2 μg/ml) for 4 h before qPCR analysis of mRNA levels of the indicated genes. (C) Knockdown of ZFYVE1 enhances EMCV- but not VSV-induced phosphorylation of TBK1, IRF3, and p65. HFFs (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi were left un-infected or infected with EMCV or VSV for the indicated times before immunoblotting analysis with the indicated antibodies. * P
    Figure Legend Snippet: Identification of ZFYVE1 as a negative regulator of MDA5-triggered signaling. (A) Knockdown of ZFYVE1 enhances transcription of downstream antiviral genes induced by EMCV but not SeV or VSV. HFFs or THP1 cells (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi plasmids were left un-infected or infected with EMCV, VSV or SeV for the indicated times (HFFs) or 6 h (THP1) before qPCR analysis of mRNA levels of the indicated genes. (B) Knockdown of ZFYVE1 enhances transcription of downstream antiviral genes induced by transfection of poly(I:C)-HMW but not poly(I:C)-LMW. HFFs (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi plasmids were mock- transfected or transfected with poly(I:C)-HMW (2 μg/ml) or poly(I:C)-LMW (2 μg/ml) for 4 h before qPCR analysis of mRNA levels of the indicated genes. (C) Knockdown of ZFYVE1 enhances EMCV- but not VSV-induced phosphorylation of TBK1, IRF3, and p65. HFFs (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi were left un-infected or infected with EMCV or VSV for the indicated times before immunoblotting analysis with the indicated antibodies. * P

    Techniques Used: Stable Transfection, Transduction, Infection, Real-time Polymerase Chain Reaction, Transfection

    Zfyve1-deficiency enhances MDA5-mediated signaling in primary mouse cells. (A) Zfyve1-deficiency enhances transcription of downstream antiviral genes induced by EMCV but not SeV or VSV in mouse cells. Zfyve1 +/+ and Zfyve1 -/- MLFs or BMDCs (2 × 10 5 ) were left un-infected or infected with EMCV, SeV or VSV for 6 h before qPCR analysis. (B) Zfyve1-deficiency increases transcription of Ifnb1 gene induced by various doses of EMCV. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-infected or infected with EMCV at MOI of 1, 3, 9 and 27 for 6 h before qPCR analysis. (C) Zfyve1-deficiency inhibits TLR3-mediated transcription of downstream genes. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-treated or treated with poly(I:C) (50 μg/ml) in the culture medium for 2 h before qPCR analysis. (D) Zfyve1-deficiency enhances transcription of downstream antiviral genes induced by transfection of poly(I:C)-HMW but not poly(I:C)-LMW in MLFs. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were transfected with lipo, poly(I:C)-LMW (2 μg/ml) or poly(I:C)-HMW (2 μg/ml) for 4 h before qPCR analysis. (E) Reconstitution of ZFYVE1 in Zfyve1 -/- inhibits EMCV-induced transcription of downstream antiviral genes. Zfyve1 -/- MLFs were reconstituted with ZFYVE1 by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with EMCV for 6 h before qPCR analysis. (F) Zfyve1-deficiency enhances EMCV- but not SeV-induced phosphorylation of TBK1, IRF3, and p65. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-infected or infected with EMCV or SeV for the indicated times before immunoblotting analysis with the indicated antibodies. * P
    Figure Legend Snippet: Zfyve1-deficiency enhances MDA5-mediated signaling in primary mouse cells. (A) Zfyve1-deficiency enhances transcription of downstream antiviral genes induced by EMCV but not SeV or VSV in mouse cells. Zfyve1 +/+ and Zfyve1 -/- MLFs or BMDCs (2 × 10 5 ) were left un-infected or infected with EMCV, SeV or VSV for 6 h before qPCR analysis. (B) Zfyve1-deficiency increases transcription of Ifnb1 gene induced by various doses of EMCV. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-infected or infected with EMCV at MOI of 1, 3, 9 and 27 for 6 h before qPCR analysis. (C) Zfyve1-deficiency inhibits TLR3-mediated transcription of downstream genes. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-treated or treated with poly(I:C) (50 μg/ml) in the culture medium for 2 h before qPCR analysis. (D) Zfyve1-deficiency enhances transcription of downstream antiviral genes induced by transfection of poly(I:C)-HMW but not poly(I:C)-LMW in MLFs. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were transfected with lipo, poly(I:C)-LMW (2 μg/ml) or poly(I:C)-HMW (2 μg/ml) for 4 h before qPCR analysis. (E) Reconstitution of ZFYVE1 in Zfyve1 -/- inhibits EMCV-induced transcription of downstream antiviral genes. Zfyve1 -/- MLFs were reconstituted with ZFYVE1 by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with EMCV for 6 h before qPCR analysis. (F) Zfyve1-deficiency enhances EMCV- but not SeV-induced phosphorylation of TBK1, IRF3, and p65. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-infected or infected with EMCV or SeV for the indicated times before immunoblotting analysis with the indicated antibodies. * P

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Transfection

    4) Product Images from "A Distinct Role of Riplet-Mediated K63-Linked Polyubiquitination of the RIG-I Repressor Domain in Human Antiviral Innate Immune Responses"

    Article Title: A Distinct Role of Riplet-Mediated K63-Linked Polyubiquitination of the RIG-I Repressor Domain in Human Antiviral Innate Immune Responses

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003533

    The association of TBK1 and IKK-ε protein kinases with RIG-I RD is enhanced by Riplet. (A–F) The interaction of RIG-I with TBK1, IKK-ε, and NEMO was examined by immunoprecipitation assay. FLAG-tagged RIG-I or RIG-I RD expression vector was transfected into HEK293FT cells together with HA-tagged IKK-ε (A, B, and E), Myc-tagged TBK1 (C, F), HA-tagged NEMO ubiquitin binding region (D), and/or Riplet (D–F) expression vectors as indicated. 24 hours after the transfection, cell lysate was prepared, and immunoprecipitation was performed with anti-FLAG antibody. Asterisk indicates non-specific bands. (G) Relative band intensity of immunoprecipitated NEMO, IKK-ε, and TBK1 in D–F was determined. (H–L) Intracellular localization of endogenous RIG-I (H–J), TBK1 (H–J), phosphorylated-TBK1 (p-TBK1) (K), and mitochondria (I–K) were observed using anti-RIG-I (Alme-1), TBK1, p-TBK1 mAbs, and mitotracker. HeLa cells were stimulated with HCV dsRNA for six hours using lipofectamine 2000. Colocalization coefficient of TBK1 localization to RIG-I (H), RIG-I and TBK1 localization to mitochondria (I), and TBK1 and p-TBK1 localization to mitochondria (L) were determined (mean ± sd, n > 10). Person's correlation coefficient of RIG-I and TBK1 was determined (H). (M) Splenocytes from wild type and Riplet KO mouse were infected with VSV at MOI = 10 for eight hours. Immunoprecipitation was performed using an anti-RIG-I rabbit monoclonal antibody (D14G6), and the immunoprecipitates were analyzed by SDS-PAGE. Endogenous RIG-I, TBK1, TRIM25, and β-actin were detected using anti-RIG-I, p-TBK1, TRIM25, and β-actin antibodies.
    Figure Legend Snippet: The association of TBK1 and IKK-ε protein kinases with RIG-I RD is enhanced by Riplet. (A–F) The interaction of RIG-I with TBK1, IKK-ε, and NEMO was examined by immunoprecipitation assay. FLAG-tagged RIG-I or RIG-I RD expression vector was transfected into HEK293FT cells together with HA-tagged IKK-ε (A, B, and E), Myc-tagged TBK1 (C, F), HA-tagged NEMO ubiquitin binding region (D), and/or Riplet (D–F) expression vectors as indicated. 24 hours after the transfection, cell lysate was prepared, and immunoprecipitation was performed with anti-FLAG antibody. Asterisk indicates non-specific bands. (G) Relative band intensity of immunoprecipitated NEMO, IKK-ε, and TBK1 in D–F was determined. (H–L) Intracellular localization of endogenous RIG-I (H–J), TBK1 (H–J), phosphorylated-TBK1 (p-TBK1) (K), and mitochondria (I–K) were observed using anti-RIG-I (Alme-1), TBK1, p-TBK1 mAbs, and mitotracker. HeLa cells were stimulated with HCV dsRNA for six hours using lipofectamine 2000. Colocalization coefficient of TBK1 localization to RIG-I (H), RIG-I and TBK1 localization to mitochondria (I), and TBK1 and p-TBK1 localization to mitochondria (L) were determined (mean ± sd, n > 10). Person's correlation coefficient of RIG-I and TBK1 was determined (H). (M) Splenocytes from wild type and Riplet KO mouse were infected with VSV at MOI = 10 for eight hours. Immunoprecipitation was performed using an anti-RIG-I rabbit monoclonal antibody (D14G6), and the immunoprecipitates were analyzed by SDS-PAGE. Endogenous RIG-I, TBK1, TRIM25, and β-actin were detected using anti-RIG-I, p-TBK1, TRIM25, and β-actin antibodies.

    Techniques Used: Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, Binding Assay, Infection, SDS Page

    Model for Riplet-mediated RIG-I activation. In resting cell, RIG-I RD represses its CARDs-mediated signaling. When RIG-I CTD associates with viral RNA, Riplet mediates K63-linked polyubiquitination of RIG-I RD, leading to the association with TRIM25 and TBK1. K63-linked polyubiquitin chain mediated by TRIM25 induces RIG-I oligomerization and association with IPS-1 adaptor. TBK1 associated with RIG-I is activated on mitochondria.
    Figure Legend Snippet: Model for Riplet-mediated RIG-I activation. In resting cell, RIG-I RD represses its CARDs-mediated signaling. When RIG-I CTD associates with viral RNA, Riplet mediates K63-linked polyubiquitination of RIG-I RD, leading to the association with TRIM25 and TBK1. K63-linked polyubiquitin chain mediated by TRIM25 induces RIG-I oligomerization and association with IPS-1 adaptor. TBK1 associated with RIG-I is activated on mitochondria.

    Techniques Used: Activation Assay

    Related Articles

    SYBR Green Assay:

    Article Title: Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination
    Article Snippet: .. Reagents and antibodies Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R & D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and d -galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies. .. HEK293 cells were obtained from ATCC.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination
    Article Snippet: .. Reagents and antibodies Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R & D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and d -galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies. .. HEK293 cells were obtained from ATCC.

    Luciferase:

    Article Title: Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination
    Article Snippet: .. Reagents and antibodies Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R & D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and d -galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies. .. HEK293 cells were obtained from ATCC.

    Article Title: Human cytomegalovirus protein UL42 antagonizes cGAS/MITA-mediated innate antiviral response
    Article Snippet: .. Reagents and antibodies 2’ 3’-cGAMP, and lipofectamine 2000 (Invitrogen); polybrene (Millipore); puromycin and RNase inhibitor (Thermo); dual-specific luciferase assay kit (Promega); SYBR (BIO-RAD); digitonin (Sigma); streptavidin agarose (Solulink); mouse antibodies against Flag, and β-actin (Sigma), and HA (Covance); rabbit monoclonal antibodies against cGAS (66546S/31659S), MITA (13647S), phosphor-MITA (85735S), phosphor-p65, and phosphor-IRF3 (4947S) (Cell Signaling Technology), phosphor-TBK1(ab109272) and TBK1(ab40676) (Abcam), IRF3 (sc-9082), phosphor-Tyrosine701-STAT1(9167S) and STAT1(sc-346) (Santa Cruz Biotechnology) were purchased from the indicated manufacturers. .. Antisera against UL42, UL82, and UL44 were generated by immunizing rabbits or mice with purified recombinant UL42, UL82, and UL44 proteins.

    Immunofluorescence:

    Article Title: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing
    Article Snippet: .. The primary antibodies used for immunofluorescence were rabbit monoclonal anti-phosphorylated TBK1 (phospho S172) (Abcam Cat# ab109272 RRID:AB_10862438, 1:500 dilution), rabbit monoclonal anti-phosphorylated TBK1 (phospho S172) (Cell Signaling Technology Cat# 5483P RRID:AB_10693472, 1:250), mouse monoclonal anti-IKKε/TBK1 (Cayman Chemical Cat# 13929 RRID:AB_10679144, 1:500), rabbit monoclonal anti-TBK1 (Abcam Cat# ab40676 RRID:AB_776632, 1:500), rabbit monoclonal anti-IRF3 (Cell Signaling Technology Cat# 4302S RRID:AB_1904036, 1:500), rabbit polyclonal anti-IRF3 (Santa Cruz Biotechnology Cat# sc-9082 RRID:AB_2264929, 1:500), mouse monoclonal anti-GM130 (BD Biosciences Cat# 610822 RRID:AB_398141, 1:1000), mouse monoclonal anti-cytochrome c (BD Biosciences Cat# 556432 RRID:AB_396416, 1:1000), mouse monoclonal anti-EEA1 (BD Biosciences Cat# 610456 RRID:AB_397829, 1:500), mouse monoclonal anti-ubiquitin (Millipore Cat# ST1200-100UG RRID:AB_2043482, 1:200), mouse monoclonal anti-giantin (Abcam Cat# ab37266 RRID:AB_880195, 1:1000), rabbit polyclonal anti-optineurin (Abcam Cat# ab23666 RRID:AB_447598, 1:500), rabbit polyclonal anti-optineurin (Cayman Chemical Cat# 100000 RRID:AB_10078198, 1:500), mouse monoclonal anti-optineurin (Santa Cruz Biotechnology Cat# sc-166576 RRID:AB_2156554, 1:500) rabbit polyclonal anti-AZI2 (NAP1) (Abcam Cat# ab65242 RRID:AB_1140792, 1:500), rabbit monoclonal anti-SINTBAD (Cell Signaling Technology Cat# 8605S RRID:AB_10839270, 1:100), rabbit polyclonal anti-giantin (Abcam Cat# ab24586 RRID:AB_448163, 1 :400), mouse monoclonal anti-mouse pericentrin (BD Biosciences Cat# 611815 RRID:AB_399295, 1:400) and a rabbit polyclonal anti-pericentrin (Abcam Cat# ab4448 RRID:AB_304461, 1:400). .. The primary antibodies used for immunoprecipitation were mouse monoclonal anti-optineurin (Santa Cruz Biotechnology Cat# sc-166576 RRID:AB_2156554), mouse monoclonal anti-GFP (Roche Cat# 11814460001 RRID:AB_390913), and rabbit monoclonal anti-TBK1 (Abcam Cat# ab40676 RRID:AB_776632).

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  • 94
    Abcam p tbk1
    Mediation of the maturation and activation of BMDCs during Mycobacterium bovis infection by the cGAS pathway. Mycobacterium bovis regulated the maturation and activation of BMDCs via the <t>cGAS/STING/TBK1/IRF3-dependent</t> pathway and promoted type I IFN production. Furthermore, type I IFN and its receptor IFNAR contributed to this process. Moreover, the mature and activated BMDCs enhanced the proliferation of T cells by some surface markers and cytokines. These signaling pathways linked the innate and adaptive immune responses. (The full line arrows signify promotion; the dotted arrows signify releasing cytokines.)
    P Tbk1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam p tbk1 antibody
    Neuropathology features of the FTD‐ALS patient with the <t>TBK1</t> p.Thr79del mutation. Severe neuronal loss, gliosis, and loosening of the neuropil are observed in entorhinal and transentorhinal region (lower image) as compared with relatively well‐preserved frontal cortex (upper image) (HE) ( A ). Abundant TDP‐43 protein aggregates in neurons and oligodendroglial cells ( B ), better seen in ( C ) at higher magnification (arrows). Hippocampal dentate gyrus shows some granular neurons lacking physiological nuclear immunoreactivity but shifting toward pathological inclusions in the cytoplasm ( D ). Morphological spectrum of neuronal cytoplasmic inclusions ( E–J ), seen as compact bodies ( E ), diffuse granular cytoplasmic immunoreactivity or “preinclusion” type ( F ), skein‐like inclusions ( G , inset), compact ring‐like inclusions ( H ), or a combination of diffuse cytoplasmic and compact in the same motor neuron ( I ). Signs of corticospinal tract degeneration at the level of the spinal cord with marked reduction of axonal density as shown by antineurofilament immunohistochemistry ( K , inset shows regular density of axons for comparison), and increased macrophagic activity ( L , anti‐CD68 immunohistochemistry, inset shows regular density of CD68+ cells in the spinal cord for comparison). Neuropathological features of concomitant argyrophilic grain pathology ( M–P ). Ballooned cells are seen in amygdala ( M ) and are nicely stained by hyperphosphorylated tau (AT8, inset). Moreover, frequent hpTau‐positive grains, mainly composed of four‐repeat tau isoforms, are detected in the limbic system ( N , CA1 sector is shown), and represent enlargements/verrucosities of dendritic spines ( N , inset). Oligodendroglial coiled bodies ( O ) and bush‐like astrocytes ( P ) accompany the full picture.
    P Tbk1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mediation of the maturation and activation of BMDCs during Mycobacterium bovis infection by the cGAS pathway. Mycobacterium bovis regulated the maturation and activation of BMDCs via the cGAS/STING/TBK1/IRF3-dependent pathway and promoted type I IFN production. Furthermore, type I IFN and its receptor IFNAR contributed to this process. Moreover, the mature and activated BMDCs enhanced the proliferation of T cells by some surface markers and cytokines. These signaling pathways linked the innate and adaptive immune responses. (The full line arrows signify promotion; the dotted arrows signify releasing cytokines.)

    Journal: International Journal of Molecular Sciences

    Article Title: cGAS/STING/TBK1/IRF3 Signaling Pathway Activates BMDCs Maturation Following Mycobacterium bovis Infection

    doi: 10.3390/ijms20040895

    Figure Lengend Snippet: Mediation of the maturation and activation of BMDCs during Mycobacterium bovis infection by the cGAS pathway. Mycobacterium bovis regulated the maturation and activation of BMDCs via the cGAS/STING/TBK1/IRF3-dependent pathway and promoted type I IFN production. Furthermore, type I IFN and its receptor IFNAR contributed to this process. Moreover, the mature and activated BMDCs enhanced the proliferation of T cells by some surface markers and cytokines. These signaling pathways linked the innate and adaptive immune responses. (The full line arrows signify promotion; the dotted arrows signify releasing cytokines.)

    Article Snippet: The antibodies used were rabbit anti-cGAS (D3080, CST, anti-MB21D1, Boston, MA, USA), rabbit anti-STING (19851-1-AP, Proteintech, Chicago, IL, USA), rabbit anti-TBK1 (ab40676, Abcam, Cambridge, UK), and p-TBK1 (ab109272, Abcam).

    Techniques: Activation Assay, Infection

    The cyclic GMP-AMP synthase (cGAS) pathway is activated in bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis infection. ( A ) BMDCs were treated with siRNA, siCon, and M. bovis , and cGAS protein was analyzed by Western blotting at 24 h after treatment. ( B ) The related proteins in the cGAS pathway in BMDCs were assayed by Western blotting. The protein levels of cGAS, p-STING, STING, TBK1, and p-TBK1 were analyzed in BMDCs transfected with siCon or sicGAS and then infected for 24 or 48 h with M. bovis (MOI 5). ( C ) The co-localization of IRF3 within the nucleus was detected by immunofluorescence microscopy (400 ×). ( D ) The culture supernatants were harvested after 24 h and assessed by ELISA. All data are expressed as mean ± SD, (* p

    Journal: International Journal of Molecular Sciences

    Article Title: cGAS/STING/TBK1/IRF3 Signaling Pathway Activates BMDCs Maturation Following Mycobacterium bovis Infection

    doi: 10.3390/ijms20040895

    Figure Lengend Snippet: The cyclic GMP-AMP synthase (cGAS) pathway is activated in bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis infection. ( A ) BMDCs were treated with siRNA, siCon, and M. bovis , and cGAS protein was analyzed by Western blotting at 24 h after treatment. ( B ) The related proteins in the cGAS pathway in BMDCs were assayed by Western blotting. The protein levels of cGAS, p-STING, STING, TBK1, and p-TBK1 were analyzed in BMDCs transfected with siCon or sicGAS and then infected for 24 or 48 h with M. bovis (MOI 5). ( C ) The co-localization of IRF3 within the nucleus was detected by immunofluorescence microscopy (400 ×). ( D ) The culture supernatants were harvested after 24 h and assessed by ELISA. All data are expressed as mean ± SD, (* p

    Article Snippet: The antibodies used were rabbit anti-cGAS (D3080, CST, anti-MB21D1, Boston, MA, USA), rabbit anti-STING (19851-1-AP, Proteintech, Chicago, IL, USA), rabbit anti-TBK1 (ab40676, Abcam, Cambridge, UK), and p-TBK1 (ab109272, Abcam).

    Techniques: Derivative Assay, Infection, Western Blot, Transfection, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay

    The EALQR motif influences SeV-mediated activation of the RIG-I signaling pathway. (A to C) The EALQR deletion in the NS1 ED weakened the inhibition of SeV-induced IFN-β, ISRE, and NF-κB promoter activities. The protocol for the luciferase reporter assay is described in Materials and Methods. The lower immunoblots show the expression levels of the transfected NS1 or NS1Δ protein. WB, Western blot. (D) The EALQR deletion in the NS1 ED impaired the inhibition of SeV-induced transcription of antiviral genes. The qPCR protocol is described in Materials and Methods. (E) The EALQR deletion in the NS1 ED decreased the inhibition of SeV-induced phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were transfected with a control, NS1, or NS1Δ plasmid. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (F) The EALQR deletion in the NS1 ED decreased the inhibition of SeV-induced phosphorylation and dimerization of IRF3. HEK293 cells were transfected with a control, NS1, or NS1Δ plasmid. Twenty-four hours after transfection, the cells were mock infected or infected with SeV for 12 h. Cell lysates were separated by native PAGE (top) or SDS-PAGE (bottom) and analyzed by immunoblotting with the indicated antibodies. (G) Effects of NS1 and NS1Δ on VSV-GFP replication. HEK293 cells were transfected with the NS1 or NS1Δ plasmid. Twenty-four hours later, the cells were infected with VSV-GFP (MOI = 0.1). The cells were viewed microscopically, and the immunoblots were analyzed with the indicated antibodies.

    Journal: Journal of Virology

    Article Title: A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity

    doi: 10.1128/JVI.00149-18

    Figure Lengend Snippet: The EALQR motif influences SeV-mediated activation of the RIG-I signaling pathway. (A to C) The EALQR deletion in the NS1 ED weakened the inhibition of SeV-induced IFN-β, ISRE, and NF-κB promoter activities. The protocol for the luciferase reporter assay is described in Materials and Methods. The lower immunoblots show the expression levels of the transfected NS1 or NS1Δ protein. WB, Western blot. (D) The EALQR deletion in the NS1 ED impaired the inhibition of SeV-induced transcription of antiviral genes. The qPCR protocol is described in Materials and Methods. (E) The EALQR deletion in the NS1 ED decreased the inhibition of SeV-induced phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were transfected with a control, NS1, or NS1Δ plasmid. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (F) The EALQR deletion in the NS1 ED decreased the inhibition of SeV-induced phosphorylation and dimerization of IRF3. HEK293 cells were transfected with a control, NS1, or NS1Δ plasmid. Twenty-four hours after transfection, the cells were mock infected or infected with SeV for 12 h. Cell lysates were separated by native PAGE (top) or SDS-PAGE (bottom) and analyzed by immunoblotting with the indicated antibodies. (G) Effects of NS1 and NS1Δ on VSV-GFP replication. HEK293 cells were transfected with the NS1 or NS1Δ plasmid. Twenty-four hours later, the cells were infected with VSV-GFP (MOI = 0.1). The cells were viewed microscopically, and the immunoblots were analyzed with the indicated antibodies.

    Article Snippet: Antibodies against TBK1 and p-TBK1 were purchased from Abcam (Cambridge, United Kingdom).

    Techniques: Activation Assay, Inhibition, Luciferase, Reporter Assay, Western Blot, Expressing, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Infection, SDS Page, Clear Native PAGE

    ELQR-4A mutations attenuate inhibition of SeV-mediated activation of the RIG-I signaling pathway. (A) The ELQR-4A mutations weakened the inhibition of SeV-induced transcription of antiviral genes. HEK293 cells were transfected with a control, NS1, or NS1-4A expression plasmid. Twenty-four hours after transfection, the cells were infected with SeV or left uninfected for 12 h before quantitative PCR was performed. (B) The ELQR-4A mutations attenuated the blockade of SeV-induced phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were transfected with a control, NS1, or NS1-4A plasmid. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (C) The ELQR-4A mutations did not inhibit RIG-I ubiquitination. HEK293 cells were transfected with the indicated plasmids for 24 h and then subjected to immunoprecipitation with anti-HA antibodies; immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-HA antibody. Whole-cell lysates were analyzed by immunoblotting with anti-Flag antibodies. (D and E) The EALQR motif is required for NS1 multimerization. HEK293 cells were cotransfected with Flag- and HA-tagged NS1 and NS1 mutants. Twenty-four hours after transfection, the cells were mock infected or infected with SeV for 18 h, and then coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies.

    Journal: Journal of Virology

    Article Title: A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity

    doi: 10.1128/JVI.00149-18

    Figure Lengend Snippet: ELQR-4A mutations attenuate inhibition of SeV-mediated activation of the RIG-I signaling pathway. (A) The ELQR-4A mutations weakened the inhibition of SeV-induced transcription of antiviral genes. HEK293 cells were transfected with a control, NS1, or NS1-4A expression plasmid. Twenty-four hours after transfection, the cells were infected with SeV or left uninfected for 12 h before quantitative PCR was performed. (B) The ELQR-4A mutations attenuated the blockade of SeV-induced phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were transfected with a control, NS1, or NS1-4A plasmid. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (C) The ELQR-4A mutations did not inhibit RIG-I ubiquitination. HEK293 cells were transfected with the indicated plasmids for 24 h and then subjected to immunoprecipitation with anti-HA antibodies; immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-HA antibody. Whole-cell lysates were analyzed by immunoblotting with anti-Flag antibodies. (D and E) The EALQR motif is required for NS1 multimerization. HEK293 cells were cotransfected with Flag- and HA-tagged NS1 and NS1 mutants. Twenty-four hours after transfection, the cells were mock infected or infected with SeV for 18 h, and then coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies.

    Article Snippet: Antibodies against TBK1 and p-TBK1 were purchased from Abcam (Cambridge, United Kingdom).

    Techniques: Inhibition, Activation Assay, Transfection, Expressing, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, SDS Page, Immunoprecipitation

    An EALQR motif in the NS1 effector domain is a key determinant for type I IFN inhibition. (A) UV-inactivated supernatants from cells infected with rescued influenza virus were added to fresh confluent A549 cells for 24 h. The cells were then infected with VSV-GFP (MOI = 0.01). GFP expression was examined under a fluorescence microscope at 12 and 24 h postinfection. (B) Schematic diagram of recombinant LaSota strains. The NS1 and NS1Δ genes were inserted between the P and M genes of the LaSota genome by using a unique PmeI restriction enzyme site. (C and D) Replication of rescued recombinant LaSota viruses. rLaSota, rLaSota-NS1, or rLaSota-NS1Δ was inoculated into 7-day-old embryonic eggs for the indicated times or used to infect A549 cells for 24 h. Viral titers were calculated as EID 50 per milliliter, or the cells were stained and analyzed with a LaSota-specific antibody. (E) Effects of two NS1 proteins on the phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were infected with rLaSota, rLaSota-NS1, or rLaSota-NS1Δ (MOI = 1) for the indicated times before immunoblot analysis.

    Journal: Journal of Virology

    Article Title: A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity

    doi: 10.1128/JVI.00149-18

    Figure Lengend Snippet: An EALQR motif in the NS1 effector domain is a key determinant for type I IFN inhibition. (A) UV-inactivated supernatants from cells infected with rescued influenza virus were added to fresh confluent A549 cells for 24 h. The cells were then infected with VSV-GFP (MOI = 0.01). GFP expression was examined under a fluorescence microscope at 12 and 24 h postinfection. (B) Schematic diagram of recombinant LaSota strains. The NS1 and NS1Δ genes were inserted between the P and M genes of the LaSota genome by using a unique PmeI restriction enzyme site. (C and D) Replication of rescued recombinant LaSota viruses. rLaSota, rLaSota-NS1, or rLaSota-NS1Δ was inoculated into 7-day-old embryonic eggs for the indicated times or used to infect A549 cells for 24 h. Viral titers were calculated as EID 50 per milliliter, or the cells were stained and analyzed with a LaSota-specific antibody. (E) Effects of two NS1 proteins on the phosphorylation of TBK1, IRF3, and IκBα. HEK293 cells were infected with rLaSota, rLaSota-NS1, or rLaSota-NS1Δ (MOI = 1) for the indicated times before immunoblot analysis.

    Article Snippet: Antibodies against TBK1 and p-TBK1 were purchased from Abcam (Cambridge, United Kingdom).

    Techniques: Inhibition, Infection, Expressing, Fluorescence, Microscopy, Recombinant, Staining

    Identification of ZFYVE1 as a negative regulator of MDA5-triggered signaling. (A) Knockdown of ZFYVE1 enhances transcription of downstream antiviral genes induced by EMCV but not SeV or VSV. HFFs or THP1 cells (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi plasmids were left un-infected or infected with EMCV, VSV or SeV for the indicated times (HFFs) or 6 h (THP1) before qPCR analysis of mRNA levels of the indicated genes. (B) Knockdown of ZFYVE1 enhances transcription of downstream antiviral genes induced by transfection of poly(I:C)-HMW but not poly(I:C)-LMW. HFFs (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi plasmids were mock- transfected or transfected with poly(I:C)-HMW (2 μg/ml) or poly(I:C)-LMW (2 μg/ml) for 4 h before qPCR analysis of mRNA levels of the indicated genes. (C) Knockdown of ZFYVE1 enhances EMCV- but not VSV-induced phosphorylation of TBK1, IRF3, and p65. HFFs (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi were left un-infected or infected with EMCV or VSV for the indicated times before immunoblotting analysis with the indicated antibodies. * P

    Journal: PLoS Pathogens

    Article Title: ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response

    doi: 10.1371/journal.ppat.1008457

    Figure Lengend Snippet: Identification of ZFYVE1 as a negative regulator of MDA5-triggered signaling. (A) Knockdown of ZFYVE1 enhances transcription of downstream antiviral genes induced by EMCV but not SeV or VSV. HFFs or THP1 cells (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi plasmids were left un-infected or infected with EMCV, VSV or SeV for the indicated times (HFFs) or 6 h (THP1) before qPCR analysis of mRNA levels of the indicated genes. (B) Knockdown of ZFYVE1 enhances transcription of downstream antiviral genes induced by transfection of poly(I:C)-HMW but not poly(I:C)-LMW. HFFs (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi plasmids were mock- transfected or transfected with poly(I:C)-HMW (2 μg/ml) or poly(I:C)-LMW (2 μg/ml) for 4 h before qPCR analysis of mRNA levels of the indicated genes. (C) Knockdown of ZFYVE1 enhances EMCV- but not VSV-induced phosphorylation of TBK1, IRF3, and p65. HFFs (1 × 10 5 ) stably transduced with control or ZFYVE1-RNAi were left un-infected or infected with EMCV or VSV for the indicated times before immunoblotting analysis with the indicated antibodies. * P

    Article Snippet: Reagents, antibodies, cells and viruses Trizol (Takara Bio), SYBR Green (Bio-Rad), RNase I (Ambion), Flag antibody-conjugated beads (Bimake), dual-specific luciferase assay kit (Promega), polybrene (Millipore), poly(I:C)-HMW (Invivogen), poly(I:C)-LMW (Invivogen), 5’ppp-dsRNA (Invivogen), DNAase I, 3 × Flag peptide (Sigma), Z-link Psoralen-PEG3-Biotin (Thermo), type II collagenase (Worthington), first-strand cDNA synthesis kit (Fermentas), GammaBind G Plus-Sepharose (Amersham Biosciences), and ELISA kits for murine IFN-β and TNF (BioLegend); mouse monoclonal antibodies for Flag and β-actin (Sigma), HA (Origene), p-IRF3, p65 and p-p65 (Cell Signaling Technology), IRF3 (Santa Cruz Biotechnology), p-TBK1 and TBK1 (Abcam), and ZFYVE1 (ABclonal); Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen); and HEK293 (American Type Culture Collection) and THP1 cells (American type culture collection) were purchased from the indicated companies.

    Techniques: Stable Transfection, Transduction, Infection, Real-time Polymerase Chain Reaction, Transfection

    Zfyve1-deficiency enhances MDA5-mediated signaling in primary mouse cells. (A) Zfyve1-deficiency enhances transcription of downstream antiviral genes induced by EMCV but not SeV or VSV in mouse cells. Zfyve1 +/+ and Zfyve1 -/- MLFs or BMDCs (2 × 10 5 ) were left un-infected or infected with EMCV, SeV or VSV for 6 h before qPCR analysis. (B) Zfyve1-deficiency increases transcription of Ifnb1 gene induced by various doses of EMCV. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-infected or infected with EMCV at MOI of 1, 3, 9 and 27 for 6 h before qPCR analysis. (C) Zfyve1-deficiency inhibits TLR3-mediated transcription of downstream genes. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-treated or treated with poly(I:C) (50 μg/ml) in the culture medium for 2 h before qPCR analysis. (D) Zfyve1-deficiency enhances transcription of downstream antiviral genes induced by transfection of poly(I:C)-HMW but not poly(I:C)-LMW in MLFs. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were transfected with lipo, poly(I:C)-LMW (2 μg/ml) or poly(I:C)-HMW (2 μg/ml) for 4 h before qPCR analysis. (E) Reconstitution of ZFYVE1 in Zfyve1 -/- inhibits EMCV-induced transcription of downstream antiviral genes. Zfyve1 -/- MLFs were reconstituted with ZFYVE1 by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with EMCV for 6 h before qPCR analysis. (F) Zfyve1-deficiency enhances EMCV- but not SeV-induced phosphorylation of TBK1, IRF3, and p65. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-infected or infected with EMCV or SeV for the indicated times before immunoblotting analysis with the indicated antibodies. * P

    Journal: PLoS Pathogens

    Article Title: ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response

    doi: 10.1371/journal.ppat.1008457

    Figure Lengend Snippet: Zfyve1-deficiency enhances MDA5-mediated signaling in primary mouse cells. (A) Zfyve1-deficiency enhances transcription of downstream antiviral genes induced by EMCV but not SeV or VSV in mouse cells. Zfyve1 +/+ and Zfyve1 -/- MLFs or BMDCs (2 × 10 5 ) were left un-infected or infected with EMCV, SeV or VSV for 6 h before qPCR analysis. (B) Zfyve1-deficiency increases transcription of Ifnb1 gene induced by various doses of EMCV. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-infected or infected with EMCV at MOI of 1, 3, 9 and 27 for 6 h before qPCR analysis. (C) Zfyve1-deficiency inhibits TLR3-mediated transcription of downstream genes. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-treated or treated with poly(I:C) (50 μg/ml) in the culture medium for 2 h before qPCR analysis. (D) Zfyve1-deficiency enhances transcription of downstream antiviral genes induced by transfection of poly(I:C)-HMW but not poly(I:C)-LMW in MLFs. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were transfected with lipo, poly(I:C)-LMW (2 μg/ml) or poly(I:C)-HMW (2 μg/ml) for 4 h before qPCR analysis. (E) Reconstitution of ZFYVE1 in Zfyve1 -/- inhibits EMCV-induced transcription of downstream antiviral genes. Zfyve1 -/- MLFs were reconstituted with ZFYVE1 by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with EMCV for 6 h before qPCR analysis. (F) Zfyve1-deficiency enhances EMCV- but not SeV-induced phosphorylation of TBK1, IRF3, and p65. Zfyve1 +/+ and Zfyve1 -/- MLFs (2 × 10 5 ) were left un-infected or infected with EMCV or SeV for the indicated times before immunoblotting analysis with the indicated antibodies. * P

    Article Snippet: Reagents, antibodies, cells and viruses Trizol (Takara Bio), SYBR Green (Bio-Rad), RNase I (Ambion), Flag antibody-conjugated beads (Bimake), dual-specific luciferase assay kit (Promega), polybrene (Millipore), poly(I:C)-HMW (Invivogen), poly(I:C)-LMW (Invivogen), 5’ppp-dsRNA (Invivogen), DNAase I, 3 × Flag peptide (Sigma), Z-link Psoralen-PEG3-Biotin (Thermo), type II collagenase (Worthington), first-strand cDNA synthesis kit (Fermentas), GammaBind G Plus-Sepharose (Amersham Biosciences), and ELISA kits for murine IFN-β and TNF (BioLegend); mouse monoclonal antibodies for Flag and β-actin (Sigma), HA (Origene), p-IRF3, p65 and p-p65 (Cell Signaling Technology), IRF3 (Santa Cruz Biotechnology), p-TBK1 and TBK1 (Abcam), and ZFYVE1 (ABclonal); Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen); and HEK293 (American Type Culture Collection) and THP1 cells (American type culture collection) were purchased from the indicated companies.

    Techniques: Infection, Real-time Polymerase Chain Reaction, Transfection

    Neuropathology features of the FTD‐ALS patient with the TBK1 p.Thr79del mutation. Severe neuronal loss, gliosis, and loosening of the neuropil are observed in entorhinal and transentorhinal region (lower image) as compared with relatively well‐preserved frontal cortex (upper image) (HE) ( A ). Abundant TDP‐43 protein aggregates in neurons and oligodendroglial cells ( B ), better seen in ( C ) at higher magnification (arrows). Hippocampal dentate gyrus shows some granular neurons lacking physiological nuclear immunoreactivity but shifting toward pathological inclusions in the cytoplasm ( D ). Morphological spectrum of neuronal cytoplasmic inclusions ( E–J ), seen as compact bodies ( E ), diffuse granular cytoplasmic immunoreactivity or “preinclusion” type ( F ), skein‐like inclusions ( G , inset), compact ring‐like inclusions ( H ), or a combination of diffuse cytoplasmic and compact in the same motor neuron ( I ). Signs of corticospinal tract degeneration at the level of the spinal cord with marked reduction of axonal density as shown by antineurofilament immunohistochemistry ( K , inset shows regular density of axons for comparison), and increased macrophagic activity ( L , anti‐CD68 immunohistochemistry, inset shows regular density of CD68+ cells in the spinal cord for comparison). Neuropathological features of concomitant argyrophilic grain pathology ( M–P ). Ballooned cells are seen in amygdala ( M ) and are nicely stained by hyperphosphorylated tau (AT8, inset). Moreover, frequent hpTau‐positive grains, mainly composed of four‐repeat tau isoforms, are detected in the limbic system ( N , CA1 sector is shown), and represent enlargements/verrucosities of dendritic spines ( N , inset). Oligodendroglial coiled bodies ( O ) and bush‐like astrocytes ( P ) accompany the full picture.

    Journal: Human Mutation

    Article Title: TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

    doi: 10.1002/humu.23161

    Figure Lengend Snippet: Neuropathology features of the FTD‐ALS patient with the TBK1 p.Thr79del mutation. Severe neuronal loss, gliosis, and loosening of the neuropil are observed in entorhinal and transentorhinal region (lower image) as compared with relatively well‐preserved frontal cortex (upper image) (HE) ( A ). Abundant TDP‐43 protein aggregates in neurons and oligodendroglial cells ( B ), better seen in ( C ) at higher magnification (arrows). Hippocampal dentate gyrus shows some granular neurons lacking physiological nuclear immunoreactivity but shifting toward pathological inclusions in the cytoplasm ( D ). Morphological spectrum of neuronal cytoplasmic inclusions ( E–J ), seen as compact bodies ( E ), diffuse granular cytoplasmic immunoreactivity or “preinclusion” type ( F ), skein‐like inclusions ( G , inset), compact ring‐like inclusions ( H ), or a combination of diffuse cytoplasmic and compact in the same motor neuron ( I ). Signs of corticospinal tract degeneration at the level of the spinal cord with marked reduction of axonal density as shown by antineurofilament immunohistochemistry ( K , inset shows regular density of axons for comparison), and increased macrophagic activity ( L , anti‐CD68 immunohistochemistry, inset shows regular density of CD68+ cells in the spinal cord for comparison). Neuropathological features of concomitant argyrophilic grain pathology ( M–P ). Ballooned cells are seen in amygdala ( M ) and are nicely stained by hyperphosphorylated tau (AT8, inset). Moreover, frequent hpTau‐positive grains, mainly composed of four‐repeat tau isoforms, are detected in the limbic system ( N , CA1 sector is shown), and represent enlargements/verrucosities of dendritic spines ( N , inset). Oligodendroglial coiled bodies ( O ) and bush‐like astrocytes ( P ) accompany the full picture.

    Article Snippet: Autophosphorylation activity of the single amino acid deletions was determined by Western blot analysis with p‐TBK1 antibody (phospho S172) (Abcam; 1:500, 84 kDa).

    Techniques: Mutagenesis, Immunohistochemistry, Activity Assay, Staining

    Impact of mutant TBK1 on NFκB activity in the IFN pathway. Graphical representation of the mean NFκB‐induced luciferase activity of identified in‐frame amino acid deletions and missense mutations found in patients‐only, shared by patients and controls, and in controls‐only, normalized to the mean signal from wild type. Luciferase activities were measured in at least three independent experiments and measured five times per experiment. The different domains are indicated in different colors as shown in the figure legend. WT, wild type TBK1 vector; Mock, empty vector containing no TBK1; S172A‐KD, p.Ser172Ala TBK1 kinase dead mutation. Mock and S172A‐KD were used as negative controls. Error bars depict standard deviation and asterisks above the bars indicate significant difference from the wild‐type level after Bonferroni correction ( P

    Journal: Human Mutation

    Article Title: TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

    doi: 10.1002/humu.23161

    Figure Lengend Snippet: Impact of mutant TBK1 on NFκB activity in the IFN pathway. Graphical representation of the mean NFκB‐induced luciferase activity of identified in‐frame amino acid deletions and missense mutations found in patients‐only, shared by patients and controls, and in controls‐only, normalized to the mean signal from wild type. Luciferase activities were measured in at least three independent experiments and measured five times per experiment. The different domains are indicated in different colors as shown in the figure legend. WT, wild type TBK1 vector; Mock, empty vector containing no TBK1; S172A‐KD, p.Ser172Ala TBK1 kinase dead mutation. Mock and S172A‐KD were used as negative controls. Error bars depict standard deviation and asterisks above the bars indicate significant difference from the wild‐type level after Bonferroni correction ( P

    Article Snippet: Autophosphorylation activity of the single amino acid deletions was determined by Western blot analysis with p‐TBK1 antibody (phospho S172) (Abcam; 1:500, 84 kDa).

    Techniques: Mutagenesis, Activity Assay, Luciferase, Plasmid Preparation, Standard Deviation

    Transcript and protein analysis of TBK1 LoF and single amino acid deletion mutations. A : gDNA and cDNA sequence traces around the c.288delT (p. Val97Phefs * 2) mutation, showing reduced expression of the mutant transcript on cDNA extracted from blood. B : gDNA and cDNA sequence traces around the c.379C > T (p.Arg127 * ) mutation, showing the absence of the mutant transcript on cDNA extracted from blood. C : Sizing of cDNA fragments generated with primers in TBK1 exon 10 and exon 13 of the c.1340+1G > A (p.Ala417 * ) carrier on cDNA extracted from blood. Sequence traces from the low‐expressed aberrant transcript demonstrates skipping of exon 11. D : Transcript and protein analysis on brain frontal cortex from the c.235_237delACA (p.Thr79del) carrier and four age‐matched control brains. The graph on the left shows the relative expression in the patient sample (blue) compared with the control samples (black) measured by quantitative real‐time PCR (qRT‐PCR). In the middle, Western blot analysis is shown of protein extracts from the patient carrier compared with control individuals. The upper band represents TBK1 (84 kDa) and the lower band represents the housekeeping protein GAPDH (37 kDa). The graph on the right shows the quantification in the patient sample (blue) and control samples (black) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the SD. E : Western blot analysis of phosphorylated TBK1 (Ser172, p‐TBK1) (upper band, 84 kDa) in HEK293T cells overexpressing the in‐frame single amino acid deletions (p.Thr79del, p.Asp167del, and p.Glu643del) compared with wild type, relative to GAPDH (lower band, 37 kDa). Mock and kinase dead (p.Ser172Ala, KD) were used as negative control. cDNA numbering according to reference sequence NM_013254.3, in addition, for intronic variants, the genomic reference sequence NC_000012.12 was used. Nucleotide positions refer to cDNA sequence and nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Protein numbering according to reference sequence NP_037386.1.

    Journal: Human Mutation

    Article Title: TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

    doi: 10.1002/humu.23161

    Figure Lengend Snippet: Transcript and protein analysis of TBK1 LoF and single amino acid deletion mutations. A : gDNA and cDNA sequence traces around the c.288delT (p. Val97Phefs * 2) mutation, showing reduced expression of the mutant transcript on cDNA extracted from blood. B : gDNA and cDNA sequence traces around the c.379C > T (p.Arg127 * ) mutation, showing the absence of the mutant transcript on cDNA extracted from blood. C : Sizing of cDNA fragments generated with primers in TBK1 exon 10 and exon 13 of the c.1340+1G > A (p.Ala417 * ) carrier on cDNA extracted from blood. Sequence traces from the low‐expressed aberrant transcript demonstrates skipping of exon 11. D : Transcript and protein analysis on brain frontal cortex from the c.235_237delACA (p.Thr79del) carrier and four age‐matched control brains. The graph on the left shows the relative expression in the patient sample (blue) compared with the control samples (black) measured by quantitative real‐time PCR (qRT‐PCR). In the middle, Western blot analysis is shown of protein extracts from the patient carrier compared with control individuals. The upper band represents TBK1 (84 kDa) and the lower band represents the housekeeping protein GAPDH (37 kDa). The graph on the right shows the quantification in the patient sample (blue) and control samples (black) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the SD. E : Western blot analysis of phosphorylated TBK1 (Ser172, p‐TBK1) (upper band, 84 kDa) in HEK293T cells overexpressing the in‐frame single amino acid deletions (p.Thr79del, p.Asp167del, and p.Glu643del) compared with wild type, relative to GAPDH (lower band, 37 kDa). Mock and kinase dead (p.Ser172Ala, KD) were used as negative control. cDNA numbering according to reference sequence NM_013254.3, in addition, for intronic variants, the genomic reference sequence NC_000012.12 was used. Nucleotide positions refer to cDNA sequence and nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Protein numbering according to reference sequence NP_037386.1.

    Article Snippet: Autophosphorylation activity of the single amino acid deletions was determined by Western blot analysis with p‐TBK1 antibody (phospho S172) (Abcam; 1:500, 84 kDa).

    Techniques: Sequencing, Mutagenesis, Expressing, Generated, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control