p tbk1 Abcam Search Results


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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 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p tbk1 antibody/product/Abcam
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    93
    Cell Signaling Technology Inc anti p tbk1
    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.
    Anti P Tbk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tbk1  (Abcam)
    92
    Abcam tbk1
    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.
    Tbk1, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam ptbk1 epr2867 2
    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.
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    92
    Abcam rabbit anti ptbk1
    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.
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    Image Search Results


    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

    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

    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