Structured Review

Abcam p tbk1
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 <t>TBK1,</t> 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.
P Tbk1, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) 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

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Recombinant:

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
Article Snippet: Antibodies against TBK1 and p-TBK1 were purchased from Abcam (Cambridge, United Kingdom). .. Antibodies against RIG-I, MDA5, and IAV NS1 were prepared in our laboratory by immunizing rabbits with the appropriate expressed recombinant proteins.

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    Abcam p tbk1
    OPTN silencing impairs <t>TBK1</t> activation after RLR or TLR3 activation. a HEK293T cells were either left unstimulated or infected with Sendai virus (SeV) for 6 and 8 h. Cell lysates (Lys.) were subjected to immunoprecipitation (IP) with an antibody against OPTN. Samples were then analyzed by immunoblotting with antibodies against the indicated proteins. b HEK293T cells were transfected with a control non-specific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN A and OPTN B) or a NEMO-specific siRNA. The cells were then transfected, 48 h later, with an IFNβ promoter reporter or with a NF-κB reporter and the Renilla luciferase gene as an internal control. Then, 24 h after transfection, cells were either left unstimulated (Unstim) or infected with Sendai virus (+ SeV) for 7 h. Luciferase assays were performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments. **** P
    P Tbk1, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OPTN silencing impairs TBK1 activation after RLR or TLR3 activation. a HEK293T cells were either left unstimulated or infected with Sendai virus (SeV) for 6 and 8 h. Cell lysates (Lys.) were subjected to immunoprecipitation (IP) with an antibody against OPTN. Samples were then analyzed by immunoblotting with antibodies against the indicated proteins. b HEK293T cells were transfected with a control non-specific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN A and OPTN B) or a NEMO-specific siRNA. The cells were then transfected, 48 h later, with an IFNβ promoter reporter or with a NF-κB reporter and the Renilla luciferase gene as an internal control. Then, 24 h after transfection, cells were either left unstimulated (Unstim) or infected with Sendai virus (+ SeV) for 7 h. Luciferase assays were performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments. **** P

    Journal: BMC Biology

    Article Title: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

    doi: 10.1186/s12915-016-0292-z

    Figure Lengend Snippet: OPTN silencing impairs TBK1 activation after RLR or TLR3 activation. a HEK293T cells were either left unstimulated or infected with Sendai virus (SeV) for 6 and 8 h. Cell lysates (Lys.) were subjected to immunoprecipitation (IP) with an antibody against OPTN. Samples were then analyzed by immunoblotting with antibodies against the indicated proteins. b HEK293T cells were transfected with a control non-specific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN A and OPTN B) or a NEMO-specific siRNA. The cells were then transfected, 48 h later, with an IFNβ promoter reporter or with a NF-κB reporter and the Renilla luciferase gene as an internal control. Then, 24 h after transfection, cells were either left unstimulated (Unstim) or infected with Sendai virus (+ SeV) for 7 h. Luciferase assays were performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments. **** P

    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).

    Techniques: Activation Assay, Infection, Immunoprecipitation, Transfection, Luciferase, Activity Assay

    Impaired TBK1 activation after RLR or TLR3 stimulation in OPTN-deficient primary cells. a Primary MEFs isolated from WT or OPTN 470T mice were infected with Sendai virus (SeV) for the indicated times. Cell lysates were then analyzed by immunoblotting with antibodies against the indicated proteins. * Indicates a non-specific band. b , c Primary MEFs isolated from WT or OPTN 470T mice were infected with SeV for the indicated times. IFNB1 and IL-6 mRNA levels were assessed by RT-qPCR with normalization against GAPDH. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT MEFs in Student’s t test). ns, not significant; AU, arbitrary unit. d , e Primary MEFs isolated from WT or OPTN 470T mice were either left unstimulated or infected with SeV for 6 or 8 h. The production of IFNβ and IL-6 was then analyzed by ELISA in the cell supernatant. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT MEFs in Student’s t test). ns, not significant. f Primary MEFs isolated from WT or OPTN 470T mice were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and aggregate counting. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). **0.001

    Journal: BMC Biology

    Article Title: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

    doi: 10.1186/s12915-016-0292-z

    Figure Lengend Snippet: Impaired TBK1 activation after RLR or TLR3 stimulation in OPTN-deficient primary cells. a Primary MEFs isolated from WT or OPTN 470T mice were infected with Sendai virus (SeV) for the indicated times. Cell lysates were then analyzed by immunoblotting with antibodies against the indicated proteins. * Indicates a non-specific band. b , c Primary MEFs isolated from WT or OPTN 470T mice were infected with SeV for the indicated times. IFNB1 and IL-6 mRNA levels were assessed by RT-qPCR with normalization against GAPDH. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT MEFs in Student’s t test). ns, not significant; AU, arbitrary unit. d , e Primary MEFs isolated from WT or OPTN 470T mice were either left unstimulated or infected with SeV for 6 or 8 h. The production of IFNβ and IL-6 was then analyzed by ELISA in the cell supernatant. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT MEFs in Student’s t test). ns, not significant. f Primary MEFs isolated from WT or OPTN 470T mice were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and aggregate counting. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). **0.001

    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).

    Techniques: Activation Assay, Isolation, Mouse Assay, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Immunofluorescence, Staining

    OPTN is targeted by the NS3 protein of the Bluetongue virus to dampen IRF3 signaling. a HeLa cells were transfected with a plasmid encoding NS3-GFP; 16 h later, the NS3-GFP localization was assessed by immunofluorescence analysis. The Golgi apparatus was stained with an antibody against GM130. Scale bars, 10 μm. b HEK293T cells were transfected with either an IFNβ promoter reporter or with a NF-κB reporter, together with the Renilla luciferase gene as an internal control. In parallel, the cells were also transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left unstimulated (Unstim) or infected with Sendai virus (+ SeV) for 7 h. Luciferase assays were then performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with GFP-transfected cells in Student’s t test). RLU, relative luminescence units. c HEK293T cells were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were infected with SeV for the indicated times. Cell lysates were analyzed by immunoblotting with antibodies against the indicated proteins. d HEK293T cells were transfected with a plasmid encoding OPTN and a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cell lysates (Lys.) were subjected to immunoprecipitation (IP) with an antibody against GFP. Samples were analyzed by immunoblotting with antibodies against the indicated proteins. e HEK293T cells were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left unstimulated or infected with Sendai virus for 7 h. Cell lysates (Lys.) were then subjected to immunoprecipitation (IP) with an antibody against OPTN. Samples were analyzed by immunoblotting with antibodies against the indicated proteins. f MEFs were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and aggregate counting in GFP-positive cells. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). *** P

    Journal: BMC Biology

    Article Title: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

    doi: 10.1186/s12915-016-0292-z

    Figure Lengend Snippet: OPTN is targeted by the NS3 protein of the Bluetongue virus to dampen IRF3 signaling. a HeLa cells were transfected with a plasmid encoding NS3-GFP; 16 h later, the NS3-GFP localization was assessed by immunofluorescence analysis. The Golgi apparatus was stained with an antibody against GM130. Scale bars, 10 μm. b HEK293T cells were transfected with either an IFNβ promoter reporter or with a NF-κB reporter, together with the Renilla luciferase gene as an internal control. In parallel, the cells were also transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left unstimulated (Unstim) or infected with Sendai virus (+ SeV) for 7 h. Luciferase assays were then performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with GFP-transfected cells in Student’s t test). RLU, relative luminescence units. c HEK293T cells were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were infected with SeV for the indicated times. Cell lysates were analyzed by immunoblotting with antibodies against the indicated proteins. d HEK293T cells were transfected with a plasmid encoding OPTN and a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cell lysates (Lys.) were subjected to immunoprecipitation (IP) with an antibody against GFP. Samples were analyzed by immunoblotting with antibodies against the indicated proteins. e HEK293T cells were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left unstimulated or infected with Sendai virus for 7 h. Cell lysates (Lys.) were then subjected to immunoprecipitation (IP) with an antibody against OPTN. Samples were analyzed by immunoblotting with antibodies against the indicated proteins. f MEFs were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and aggregate counting in GFP-positive cells. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). *** P

    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).

    Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining, Luciferase, Infection, Activity Assay, Immunoprecipitation

    Localization of the active form of TBK1 at the Golgi apparatus after RLR stimulation. a MEFs were either left unstimulated or infected with Sendai virus (SeV) for 6 or 8 h. MEFs were then fractionated as described in Additional file 1 A, and samples were analyzed by immunoblotting with antibodies against the indicated proteins. EEA1, kinectin, LAMP2, GAPDH, syntaxin-6, and VDAC served as loading and purity controls for endosomes, the endoplasmic reticulum, lysosomes, the cytosol, the Golgi apparatus, and mitochondria, respectively. (Ub)n, polyubiquitin. * Indicates non-specific bands. b – e MEFs were either left unstimulated (control) or infected with SeV for 6 h (+ SeV). The indicated proteins were then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody raised against GM130, whereas the mitochondria were identified by labeling with an antibody against cytochrome c. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. f WT or TBK1 –/– MEFs were either left unstimulated (control) or infected with SeV for 6 h (+ SeV). p-TBK1 S172 staining was then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody raised against GM130. Scale bars, 10 μm. g Crude heavy membrane fractions from uninfected or SeV-infected MEFs were fractionated on OptiPrep density gradients (fractions range from 1 at the top to 4 at the bottom) and analyzed by immunoblotting with antibodies against the indicated proteins. (Ub)n, polyubiquitin. h Increased concentrations of mitochondria (P5) or Golgi (P25)-enriched fractions of unstimulated (Unst) or SeV-infected HEK293T cells were incubated with recombinant GST-IRF3 in the presence of ATP. The degree of IRF3 phosphorylation was determined by immunoblotting

    Journal: BMC Biology

    Article Title: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

    doi: 10.1186/s12915-016-0292-z

    Figure Lengend Snippet: Localization of the active form of TBK1 at the Golgi apparatus after RLR stimulation. a MEFs were either left unstimulated or infected with Sendai virus (SeV) for 6 or 8 h. MEFs were then fractionated as described in Additional file 1 A, and samples were analyzed by immunoblotting with antibodies against the indicated proteins. EEA1, kinectin, LAMP2, GAPDH, syntaxin-6, and VDAC served as loading and purity controls for endosomes, the endoplasmic reticulum, lysosomes, the cytosol, the Golgi apparatus, and mitochondria, respectively. (Ub)n, polyubiquitin. * Indicates non-specific bands. b – e MEFs were either left unstimulated (control) or infected with SeV for 6 h (+ SeV). The indicated proteins were then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody raised against GM130, whereas the mitochondria were identified by labeling with an antibody against cytochrome c. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. f WT or TBK1 –/– MEFs were either left unstimulated (control) or infected with SeV for 6 h (+ SeV). p-TBK1 S172 staining was then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody raised against GM130. Scale bars, 10 μm. g Crude heavy membrane fractions from uninfected or SeV-infected MEFs were fractionated on OptiPrep density gradients (fractions range from 1 at the top to 4 at the bottom) and analyzed by immunoblotting with antibodies against the indicated proteins. (Ub)n, polyubiquitin. h Increased concentrations of mitochondria (P5) or Golgi (P25)-enriched fractions of unstimulated (Unst) or SeV-infected HEK293T cells were incubated with recombinant GST-IRF3 in the presence of ATP. The degree of IRF3 phosphorylation was determined by immunoblotting

    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).

    Techniques: Infection, Immunofluorescence, Staining, Labeling, Incubation, Recombinant

    The active form of TBK1 localizes at the Golgi apparatus after TLR3 stimulation. a HEK293T cells stably expressing HA-TLR3 were either left untreated or stimulated with poly(I:C) (10 μg/mL) for 30 or 60 minutes. Cells were then fractionated as described in Additional file 1 A, and samples were analyzed by immunoblotting with antibodies against the indicated proteins. EEA1, kinectin, LAMP2, GAPDH, syntaxin-6, and VDAC served as loading and purity controls for endosomes, the endoplasmic reticulum, lysosomes, the cytosol, the Golgi apparatus, and mitochondria, respectively. (Ub)n, polyubiquitin. * Indicates non-specific bands. b , c HEK293T cells stably expressing HA-TLR3 were either left untreated (control) or stimulated with poly(I:C) (10 μg/mL) for 1 h (+ Poly(I:C)). The indicated proteins were then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody against GM130, and an antibody against EEA1 was used to stain endosomes. Scale bars, 10 μm. In b, on the right, enlargement of the framed zone in the overlay. d Crude heavy membrane fractions from unstimulated or poly(I:C)-treated HEK293T cells stably expressing HA-TLR3 were fractionated on OptiPrep density gradients (fractions range from 1 at the top to 4 at the bottom) and analyzed by immunoblotting with antibodies against the indicated proteins. (Ub)n, polyubiquitin. * Indicates non-specific bands. e Increased concentrations of mitochondria- (P5) or Golgi (P25)-enriched fractions of unstimulated (Unst) or poly(I:C)-treated HEK293T cells stably expressing HA-TLR3 were incubated with recombinant GST-IRF3 in the presence of ATP. The degree of IRF3 phosphorylation was determined by immunoblotting

    Journal: BMC Biology

    Article Title: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

    doi: 10.1186/s12915-016-0292-z

    Figure Lengend Snippet: The active form of TBK1 localizes at the Golgi apparatus after TLR3 stimulation. a HEK293T cells stably expressing HA-TLR3 were either left untreated or stimulated with poly(I:C) (10 μg/mL) for 30 or 60 minutes. Cells were then fractionated as described in Additional file 1 A, and samples were analyzed by immunoblotting with antibodies against the indicated proteins. EEA1, kinectin, LAMP2, GAPDH, syntaxin-6, and VDAC served as loading and purity controls for endosomes, the endoplasmic reticulum, lysosomes, the cytosol, the Golgi apparatus, and mitochondria, respectively. (Ub)n, polyubiquitin. * Indicates non-specific bands. b , c HEK293T cells stably expressing HA-TLR3 were either left untreated (control) or stimulated with poly(I:C) (10 μg/mL) for 1 h (+ Poly(I:C)). The indicated proteins were then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody against GM130, and an antibody against EEA1 was used to stain endosomes. Scale bars, 10 μm. In b, on the right, enlargement of the framed zone in the overlay. d Crude heavy membrane fractions from unstimulated or poly(I:C)-treated HEK293T cells stably expressing HA-TLR3 were fractionated on OptiPrep density gradients (fractions range from 1 at the top to 4 at the bottom) and analyzed by immunoblotting with antibodies against the indicated proteins. (Ub)n, polyubiquitin. * Indicates non-specific bands. e Increased concentrations of mitochondria- (P5) or Golgi (P25)-enriched fractions of unstimulated (Unst) or poly(I:C)-treated HEK293T cells stably expressing HA-TLR3 were incubated with recombinant GST-IRF3 in the presence of ATP. The degree of IRF3 phosphorylation was determined by immunoblotting

    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).

    Techniques: Stable Transfection, Expressing, Immunofluorescence, Staining, Incubation, Recombinant

    Ubiquitination promotes TBK1 targeting to the Golgi apparatus for activation. a HEK293T cells were transfected with an empty vector (Ev) or with plasmids encoding myc-tagged WT TBK1 (WT), TBK1 K38M (K38M), or TBK1 K30R/K401R (K30R/K401R). After 16 h, TBK1 activation and exogenous TBK1 expression were assessed by immunoblotting with anti-p-TBK1 S172 and anti-myc antibodies, respectively. GAPDH was used as a loading control. b HEK293T cells were transfected with either an IFNβ promoter reporter or an NF-κB reporter, together with the Renilla luciferase gene as an internal control. In parallel, the cells were also transfected with an Ev or with plasmids encoding myc-tagged WT TBK1 (WT), TBK1 K38M (K38M), or TBK1 K30R/K401R (K30R/K401R). Luciferase assays were performed 24 h after transfection and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT TBK1 in Student’s t test). RLU, relative luminescence units. c Immunoblotting analysis of TBK1 –/– MEFs reconstituted with WT TBK1, TBK1 K38M (K38M), or TBK1 K30R/K401R (K30R/K401R). As controls, TBK1 +/+ and TBK1 –/– MEFs are shown. d TBK1 –/– MEFs reconstituted with WT TBK1 or mutants were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was then assessed by immunofluorescence staining and counting of the aggregates. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). **0.001

    Journal: BMC Biology

    Article Title: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

    doi: 10.1186/s12915-016-0292-z

    Figure Lengend Snippet: Ubiquitination promotes TBK1 targeting to the Golgi apparatus for activation. a HEK293T cells were transfected with an empty vector (Ev) or with plasmids encoding myc-tagged WT TBK1 (WT), TBK1 K38M (K38M), or TBK1 K30R/K401R (K30R/K401R). After 16 h, TBK1 activation and exogenous TBK1 expression were assessed by immunoblotting with anti-p-TBK1 S172 and anti-myc antibodies, respectively. GAPDH was used as a loading control. b HEK293T cells were transfected with either an IFNβ promoter reporter or an NF-κB reporter, together with the Renilla luciferase gene as an internal control. In parallel, the cells were also transfected with an Ev or with plasmids encoding myc-tagged WT TBK1 (WT), TBK1 K38M (K38M), or TBK1 K30R/K401R (K30R/K401R). Luciferase assays were performed 24 h after transfection and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT TBK1 in Student’s t test). RLU, relative luminescence units. c Immunoblotting analysis of TBK1 –/– MEFs reconstituted with WT TBK1, TBK1 K38M (K38M), or TBK1 K30R/K401R (K30R/K401R). As controls, TBK1 +/+ and TBK1 –/– MEFs are shown. d TBK1 –/– MEFs reconstituted with WT TBK1 or mutants were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was then assessed by immunofluorescence staining and counting of the aggregates. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). **0.001

    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).

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Immunofluorescence, Staining

    TBK1 forms aggregates at Golgi apparatus following potent RLR activation. a MEFs were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 2 h (trPoly(I:C)). TBK1 staining was then analyzed by immunofluorescence. The Golgi apparatus was identified by labeling with an antibody raised against GM130. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. b WT or TBK1 –/– MEFs were stained with an antibody raised against TBK1, then analyzed by immunofluorescence analysis. c MEFs were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 2 h (trPoly(I:C)). The indicated proteins were analyzed by immunofluorescence analysis with specific antibodies. Scale bars, 10 μm. d WT, MAVS –/– , or STING –/– MEFs were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and aggregate counting. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). *** P

    Journal: BMC Biology

    Article Title: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

    doi: 10.1186/s12915-016-0292-z

    Figure Lengend Snippet: TBK1 forms aggregates at Golgi apparatus following potent RLR activation. a MEFs were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 2 h (trPoly(I:C)). TBK1 staining was then analyzed by immunofluorescence. The Golgi apparatus was identified by labeling with an antibody raised against GM130. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. b WT or TBK1 –/– MEFs were stained with an antibody raised against TBK1, then analyzed by immunofluorescence analysis. c MEFs were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 2 h (trPoly(I:C)). The indicated proteins were analyzed by immunofluorescence analysis with specific antibodies. Scale bars, 10 μm. d WT, MAVS –/– , or STING –/– MEFs were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and aggregate counting. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). *** P

    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).

    Techniques: Activation Assay, Transfection, Staining, Immunofluorescence, Labeling

    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