rabbit monoclonal antibodies against tlr3 Cell Signaling Technology Inc Search Results


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    Cell Signaling Technology Inc rabbit antibodies against p irf3
    Rabbit Antibodies Against P Irf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 81/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gapdh
    Effect of PEF on the activation of TLRs, MAPKs, and NF- κ B in APAP-intoxicated mouse livers. (a) Total protein was extracted from liver tissues, and the protein levels of TLR3, TLR4, <t>p-JNK,</t> JNK, p-ERK, ERK, p-p38, p-38, p-IKK α / β , IKK α / β , p-I κ B- α , I κ B- α , p-NF- κ B, and NF- κ B were determined by Western blot analysis. <t>GAPDH</t> was used as an endogenous control. (b) Total RNA was isolated from liver tissues and reverse-transcribed into cDNA for RT-PCR analysis of TLR3 and TLR4 mRNA level. GAPDH was used as an endogenous control. Results are shown as the mean ± SD ( n = 8). The different letters represent the statistical differences at p
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 24083 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc myc
    Effect of PEF on the activation of TLRs, MAPKs, and NF- κ B in APAP-intoxicated mouse livers. (a) Total protein was extracted from liver tissues, and the protein levels of TLR3, TLR4, <t>p-JNK,</t> JNK, p-ERK, ERK, p-p38, p-38, p-IKK α / β , IKK α / β , p-I κ B- α , I κ B- α , p-NF- κ B, and NF- κ B were determined by Western blot analysis. <t>GAPDH</t> was used as an endogenous control. (b) Total RNA was isolated from liver tissues and reverse-transcribed into cDNA for RT-PCR analysis of TLR3 and TLR4 mRNA level. GAPDH was used as an endogenous control. Results are shown as the mean ± SD ( n = 8). The different letters represent the statistical differences at p
    Myc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc runx3
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Runx3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc runx2
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
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    Cell Signaling Technology Inc desmin
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Desmin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rig i
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Rig I, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 11865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gsk3β
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
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    Cell Signaling Technology Inc alexafluor 647 mouse monoclonal antibody igg1 isotype control
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Alexafluor 647 Mouse Monoclonal Antibody Igg1 Isotype Control, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho ikkα β
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Phospho Ikkα β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 572 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mab anti p62 sqstm1 mbl
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
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    Cell Signaling Technology Inc antibodies against phospho p53
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Antibodies Against Phospho P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cleaved parp
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Cleaved Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho ibα
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    Phospho Ibα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 76/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p iκbα
    <t>Runx3</t> mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).
    P Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho irf3
    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of <t>IRF3</t> and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P
    Phospho Irf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc tlr3
    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of <t>IRF3</t> and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P
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    Cell Signaling Technology Inc p p65
    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of <t>IRF3</t> and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P
    P P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2082 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc alexa fluor 647 mouse anti glial fibrillary acidic protein gfap
    Analysis of TLR4, CD14 and MD2 expression in enriched and L-LME-purified astrocytes. a Western blots show comparable levels of TLR4, CD14 and MD2 in both astrocyte populations. Left panel : representative immunoblot. Right panel : Mean values from duplicate samples. b Confocal microscopy shows co-expression of <t>GFAP</t> ( green ) and TLR4 ( red ) in enriched (L-LME - ) as well as in purified (L-LME + ) astrocytes. Lower frame in each case illustrates GFAP/TLR4 overlap. Nuclei are labelled with DAPI ( blue ). c LPS conjugated with <t>Alexa</t> Fluor® 594 ( red ) co-localizes with GFAP ( green ) in L-LME-purified astrocytes after 30 min of treatment ( arrow ). Nuclei are labelled with DAPI
    Alexa Fluor 647 Mouse Anti Glial Fibrillary Acidic Protein Gfap, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc caspase 3
    MT decreases the expression of TLR-4, MyD88 and <t>caspase-3</t> proteins in LPS-stimulated BV2 cells. BV2 microglia cells were stimulated with 1 µg with 1 µg/ml LPS for 30 min, followed by treatment with 7.5 µg/ml MT for 24 h prior to
    Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 20177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MT decreases the expression of TLR-4, <t>MyD88</t> and caspase-3 proteins in LPS-stimulated BV2 cells. BV2 microglia cells were stimulated with 1 µg with 1 µg/ml LPS for 30 min, followed by treatment with 7.5 µg/ml MT for 24 h prior to
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    Investigation of leptin receptor expression and signalling pathways leading to MMP-1 synthesis in HGFs. Flow cytometry for cell surface leptin receptor expression on unstimulated HGFs (A). Data are representative of similar data from 4 HGF donors. Inset (A): long LEPR isoform (Ob-Rb) and β2m gene expression as assessed by RT-PCR in unstimulated HGFs. Data are representative of similar results from 7 HGF donors. HGFs were stimulated with leptin (10 μg/ml), IL-1 (0.05 ng/ml), pam2CSK4 (50 ng/ml), OSM (5 ng/ml), leptin+IL-1 or leptin+pam2CSK4 for 20 min and lysates immunoblotted with the indicated antibodies including GAPDH as a loading control (B). The blots presented are all derived from the same donor and are representative of similar results from 3 donors stimulated in independent experiments. (C) HGFs were pre-treated with the ERK inhibitor U0126 (7.5 μM), (D) the JNK inhibitor SP600126 (10 μM), (E) <t>STAT3</t> inhibitor VI (100 μM), or (F) the p38 inhibitor SB203580 (10 μM) for 30 min and then stimulated with leptin (10 μg/ml), IL-1α (0.05 ng/ml), pam2CSK4 (50 ng/ml), leptin+IL-1α or leptin+pam2CSK4 for 24 h. MMP-1 gene expression was assessed by real-time RT-PCR. Data (fold unstimulated DMSO-pre-treated control) are expressed relative to RNAP. Data are presented as mean+SEM of three donors stimulated in independent experiments (n = 4 for each donor). * P
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    Investigation of leptin receptor expression and signalling pathways leading to MMP-1 synthesis in HGFs. Flow cytometry for cell surface leptin receptor expression on unstimulated HGFs (A). Data are representative of similar data from 4 HGF donors. Inset (A): long LEPR isoform (Ob-Rb) and β2m gene expression as assessed by RT-PCR in unstimulated HGFs. Data are representative of similar results from 7 HGF donors. HGFs were stimulated with leptin (10 μg/ml), IL-1 (0.05 ng/ml), pam2CSK4 (50 ng/ml), OSM (5 ng/ml), leptin+IL-1 or leptin+pam2CSK4 for 20 min and lysates immunoblotted with the indicated antibodies including GAPDH as a loading control (B). The blots presented are all derived from the same donor and are representative of similar results from 3 donors stimulated in independent experiments. (C) HGFs were pre-treated with the ERK inhibitor U0126 (7.5 μM), (D) the JNK inhibitor SP600126 (10 μM), (E) <t>STAT3</t> inhibitor VI (100 μM), or (F) the p38 inhibitor SB203580 (10 μM) for 30 min and then stimulated with leptin (10 μg/ml), IL-1α (0.05 ng/ml), pam2CSK4 (50 ng/ml), leptin+IL-1α or leptin+pam2CSK4 for 24 h. MMP-1 gene expression was assessed by real-time RT-PCR. Data (fold unstimulated DMSO-pre-treated control) are expressed relative to RNAP. Data are presented as mean+SEM of three donors stimulated in independent experiments (n = 4 for each donor). * P
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    Santa Cruz Biotechnology toll like receptor 4 tlr 4
    Investigation of leptin receptor expression and signalling pathways leading to MMP-1 synthesis in HGFs. Flow cytometry for cell surface leptin receptor expression on unstimulated HGFs (A). Data are representative of similar data from 4 HGF donors. Inset (A): long LEPR isoform (Ob-Rb) and β2m gene expression as assessed by RT-PCR in unstimulated HGFs. Data are representative of similar results from 7 HGF donors. HGFs were stimulated with leptin (10 μg/ml), IL-1 (0.05 ng/ml), pam2CSK4 (50 ng/ml), OSM (5 ng/ml), leptin+IL-1 or leptin+pam2CSK4 for 20 min and lysates immunoblotted with the indicated antibodies including GAPDH as a loading control (B). The blots presented are all derived from the same donor and are representative of similar results from 3 donors stimulated in independent experiments. (C) HGFs were pre-treated with the ERK inhibitor U0126 (7.5 μM), (D) the JNK inhibitor SP600126 (10 μM), (E) <t>STAT3</t> inhibitor VI (100 μM), or (F) the p38 inhibitor SB203580 (10 μM) for 30 min and then stimulated with leptin (10 μg/ml), IL-1α (0.05 ng/ml), pam2CSK4 (50 ng/ml), leptin+IL-1α or leptin+pam2CSK4 for 24 h. MMP-1 gene expression was assessed by real-time RT-PCR. Data (fold unstimulated DMSO-pre-treated control) are expressed relative to RNAP. Data are presented as mean+SEM of three donors stimulated in independent experiments (n = 4 for each donor). * P
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    <t>Angiogenin,</t> Angiopoietin-1, TLR4, NFkB and RAGE expression in the ischemic brain
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    Image Search Results


    Effect of PEF on the activation of TLRs, MAPKs, and NF- κ B in APAP-intoxicated mouse livers. (a) Total protein was extracted from liver tissues, and the protein levels of TLR3, TLR4, p-JNK, JNK, p-ERK, ERK, p-p38, p-38, p-IKK α / β , IKK α / β , p-I κ B- α , I κ B- α , p-NF- κ B, and NF- κ B were determined by Western blot analysis. GAPDH was used as an endogenous control. (b) Total RNA was isolated from liver tissues and reverse-transcribed into cDNA for RT-PCR analysis of TLR3 and TLR4 mRNA level. GAPDH was used as an endogenous control. Results are shown as the mean ± SD ( n = 8). The different letters represent the statistical differences at p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Anti-Inflammatory Effect of a Polyphenol-Enriched Fraction from Acalypha wilkesiana on Lipopolysaccharide-Stimulated RAW 264.7 Macrophages and Acetaminophen-Induced Liver Injury in Mice

    doi: 10.1155/2018/7858094

    Figure Lengend Snippet: Effect of PEF on the activation of TLRs, MAPKs, and NF- κ B in APAP-intoxicated mouse livers. (a) Total protein was extracted from liver tissues, and the protein levels of TLR3, TLR4, p-JNK, JNK, p-ERK, ERK, p-p38, p-38, p-IKK α / β , IKK α / β , p-I κ B- α , I κ B- α , p-NF- κ B, and NF- κ B were determined by Western blot analysis. GAPDH was used as an endogenous control. (b) Total RNA was isolated from liver tissues and reverse-transcribed into cDNA for RT-PCR analysis of TLR3 and TLR4 mRNA level. GAPDH was used as an endogenous control. Results are shown as the mean ± SD ( n = 8). The different letters represent the statistical differences at p

    Article Snippet: Antibodies against iNOS (13120), IKKα (11930), IKKβ (8943), phospho-IKKα /β (2697), NF-κ B p65 (8242), phospho-NF-κ B p65 (3033), Iκ B-α (4814), phospho-Iκ B-α (2859), JNK (9252), phospho-JNK (4668), ERK1/2 (4695), phospho-ERK1/2 (4370), p38 (8690), phospho-p38 (4511), and GAPDH (2118) were purchased from Cell Signaling Technology (Beverly, MA, USA), antibodies against TLR3 (ab62566) and TLR4 (ab13556) were purchased from Abcam (Cambridge, UK), and antibody against COX-2 (A1253) was purchased from ABclonal Biotechnology (Wuhan, China).

    Techniques: Activation Assay, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of PEF on LPS-stimulated phosphorylation of MAPKs in RAW 264.7 macrophages. (a) RAW 264.7 macrophages were preincubated with 200 μ g/mL PEF for 2 h, and then, the total protein was harvested at different time points (10, 15, 30, and 60 min) after stimulation with LPS (1 μ g/mL), and the protein levels of p-ERK, ERK, p-p38, p38, p-JNK, and JNK were determined by Western blot analysis. GAPDH was used as an endogenous control. (b) Relative intensity of the immunoreactive bands was analyzed, and results are shown as the mean ± SD ( n = 3). Statistical difference between two groups in the presence and absence of PEF was evaluated by Student's t -test. a p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Anti-Inflammatory Effect of a Polyphenol-Enriched Fraction from Acalypha wilkesiana on Lipopolysaccharide-Stimulated RAW 264.7 Macrophages and Acetaminophen-Induced Liver Injury in Mice

    doi: 10.1155/2018/7858094

    Figure Lengend Snippet: Effect of PEF on LPS-stimulated phosphorylation of MAPKs in RAW 264.7 macrophages. (a) RAW 264.7 macrophages were preincubated with 200 μ g/mL PEF for 2 h, and then, the total protein was harvested at different time points (10, 15, 30, and 60 min) after stimulation with LPS (1 μ g/mL), and the protein levels of p-ERK, ERK, p-p38, p38, p-JNK, and JNK were determined by Western blot analysis. GAPDH was used as an endogenous control. (b) Relative intensity of the immunoreactive bands was analyzed, and results are shown as the mean ± SD ( n = 3). Statistical difference between two groups in the presence and absence of PEF was evaluated by Student's t -test. a p

    Article Snippet: Antibodies against iNOS (13120), IKKα (11930), IKKβ (8943), phospho-IKKα /β (2697), NF-κ B p65 (8242), phospho-NF-κ B p65 (3033), Iκ B-α (4814), phospho-Iκ B-α (2859), JNK (9252), phospho-JNK (4668), ERK1/2 (4695), phospho-ERK1/2 (4370), p38 (8690), phospho-p38 (4511), and GAPDH (2118) were purchased from Cell Signaling Technology (Beverly, MA, USA), antibodies against TLR3 (ab62566) and TLR4 (ab13556) were purchased from Abcam (Cambridge, UK), and antibody against COX-2 (A1253) was purchased from ABclonal Biotechnology (Wuhan, China).

    Techniques: Western Blot

    Runx3 mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: Runx3 mRNA is induced by dsRNA poly(I:C) via transcriptional regulation but not mRNA stability. ( a ) BEAS-2B cells were treated with control PBS, low molecular weight poly(I:C) (PIC-L, 10 μg/ml), or high molecular weight poly(I:C) (PIC-H, 10 μg/ml) for 16 h ( left panels ); or pretreated 2 h with (+) or without (–) actinomycin D (Act.D, 5 μM) followed by incubation with high molecular weight poly(I:C) (PIC, 10 μg/ml) for 16 h ( right panels ); and Runx3 mRNA levels are shown in a representative RT-PCR of three independent experiments. ( b ) BEAS-2B cells were treated with control PBS or high molecular weight poly(I:C) (5 μg/ml) for 16 h, then actinomycin D (Act.D, 5 μM) was added for 3 or 6 h. Relative change in Runx3 mRNA level was measured and normalized to GAPDH and is represented as percentage of Runx3 mRNA at 0 h (n = 3).

    Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.

    Techniques: Molecular Weight, Activated Clotting Time Assay, Incubation, Reverse Transcription Polymerase Chain Reaction

    Both the extrinsic and intrinsic pathways are involved in Runx3-promoted cell apoptosis by dsRNA poly(I:C) and IAV infection. ( a ) Runx3 is required for the activation of caspase-8 and caspsas-9 by poly(I:C). BEAS-2B cells were transfected with 20 nM control siRNA (Con.), Runx3 siRNA-1 (Runx3-S1), or Runx3 siRNA-2 (Runx3-S2), grown for 72 h, then treated with high molecular weight poly(I:C) (2 μg/ml) for 0, 2 or 4 h. Equal amounts of cell lysates were subjected to Western blotting with specific antibodies against cleaved caspase-8, cleaved caspase-9, Runx3 or action as indicated. ( b,c ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone or Runx3 and grown for 60 h. The cells were then left untreated or treated with poly(I:C) (2 μg/ml) for 4 h ( b ) or infected with (+) IAV H1N1 at MOI of 3 for 24 h ( c ) in the presence or absence of caspase-8 inhibitor Z-IETD-FMK (IETD-8, 20 μM), caspace-9 inhibitor Z-LEHD-FMK (LEHD-9, 20 μM), or a general caspase inhibitor Z-VAD-FMK (Z-VAD, 20 μM), and cell death rate was assessed as in Figure 7 . All data are means ± S.E. of triplicates. *p

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: Both the extrinsic and intrinsic pathways are involved in Runx3-promoted cell apoptosis by dsRNA poly(I:C) and IAV infection. ( a ) Runx3 is required for the activation of caspase-8 and caspsas-9 by poly(I:C). BEAS-2B cells were transfected with 20 nM control siRNA (Con.), Runx3 siRNA-1 (Runx3-S1), or Runx3 siRNA-2 (Runx3-S2), grown for 72 h, then treated with high molecular weight poly(I:C) (2 μg/ml) for 0, 2 or 4 h. Equal amounts of cell lysates were subjected to Western blotting with specific antibodies against cleaved caspase-8, cleaved caspase-9, Runx3 or action as indicated. ( b,c ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone or Runx3 and grown for 60 h. The cells were then left untreated or treated with poly(I:C) (2 μg/ml) for 4 h ( b ) or infected with (+) IAV H1N1 at MOI of 3 for 24 h ( c ) in the presence or absence of caspase-8 inhibitor Z-IETD-FMK (IETD-8, 20 μM), caspace-9 inhibitor Z-LEHD-FMK (LEHD-9, 20 μM), or a general caspase inhibitor Z-VAD-FMK (Z-VAD, 20 μM), and cell death rate was assessed as in Figure 7 . All data are means ± S.E. of triplicates. *p

    Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.

    Techniques: Infection, Activation Assay, Transfection, Molecular Weight, Western Blot, Recombinant, Plasmid Preparation

    Runx3 is strongly induced by dsRNA poly(I:C) in airway epithelial cells. ( a,b ) SAECs ( a ) and BEAS-2B ( b ) cells were treated 24 h with control PBS (control), low molecular weight poly(I:C) (PIC-L, 10 μg/ml), high molecular weight poly(I:C) (PIC-H, 10 μg/ml), R837 (10 μg/ml), CL075 (10 μg/ml), TGFβ (10 ng/ml), TNFα (6 ng/ml), CpG (2 μg/ml), LPS (1 μg/ml), or Pam3CSK4 (Pam3, 10 μg/ml) as indicated. ( c ) BEAS-2B cells were treated 24 h with different doses of a high molecular weight poly(I:C) (PIC-H) ( left panels ) or with the poly(I:C) (5 μg/ml) for 0-24 h ( right panels ). ( d ) BEAS-2B cells were treated 24 h with different doses of a high molecular weight poly(I:C) complexed with (+) or without (–) lipofectamine-2000. Cell lysates at equal protein amounts from ( a–d ) were subjected to Western blot analysis with Runx3 or actin antibodies. Results shown are representative Western blots of three independent experiments. ( e ) BEAS-2B cells were treated with control PBS or poly(I:C) (2 μg/ml) for 20 h, fixed and immunostained with Runx3 monoclonal antibody or stained with DAPI as indicated. Nuclear localization of Runx3 was visualized and photographed by fluorescence microscopy. Results represent the findings of two independent experiments.

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: Runx3 is strongly induced by dsRNA poly(I:C) in airway epithelial cells. ( a,b ) SAECs ( a ) and BEAS-2B ( b ) cells were treated 24 h with control PBS (control), low molecular weight poly(I:C) (PIC-L, 10 μg/ml), high molecular weight poly(I:C) (PIC-H, 10 μg/ml), R837 (10 μg/ml), CL075 (10 μg/ml), TGFβ (10 ng/ml), TNFα (6 ng/ml), CpG (2 μg/ml), LPS (1 μg/ml), or Pam3CSK4 (Pam3, 10 μg/ml) as indicated. ( c ) BEAS-2B cells were treated 24 h with different doses of a high molecular weight poly(I:C) (PIC-H) ( left panels ) or with the poly(I:C) (5 μg/ml) for 0-24 h ( right panels ). ( d ) BEAS-2B cells were treated 24 h with different doses of a high molecular weight poly(I:C) complexed with (+) or without (–) lipofectamine-2000. Cell lysates at equal protein amounts from ( a–d ) were subjected to Western blot analysis with Runx3 or actin antibodies. Results shown are representative Western blots of three independent experiments. ( e ) BEAS-2B cells were treated with control PBS or poly(I:C) (2 μg/ml) for 20 h, fixed and immunostained with Runx3 monoclonal antibody or stained with DAPI as indicated. Nuclear localization of Runx3 was visualized and photographed by fluorescence microscopy. Results represent the findings of two independent experiments.

    Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.

    Techniques: Molecular Weight, Western Blot, Staining, Fluorescence, Microscopy

    IAV-induced expression of Runx3 is primarily mediated by MDA5−NF-κB pathway. ( a , b ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con), human MDA5 siRNA-2 (M), TLR3 siRNA-2 (T), both MDA5 and TLR3 siRNAs (M + T), RIG-1 siRNA-1 (S1), or RIG-1 siRNA-2 (S2), grown for 72 h then infected without (−) or with (+) H1N1 PR/8/34 at MOI of 1 for 24 h. ( c,d ) BEAS-2B cells were transfected with control H 2 O (Con), or different amounts of RNA isolated from uninfected BEAS-2B cells (C-RNA) or H1N1-infected BEAS-2B cells (V-RNA) treated with or without calf intestine alkaline phosphatase (CIAP). After 24 h, IFNβ released into the medium was measured by ELISA ( c ) or equal amounts of cell lysates were subjected to Western blot analysis ( d ). ( e )BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con) or human MDA5 siRNA-2 (M), grown for 72 h, then transfected with control H 2 O (Control) or total RNA isolated from H1N1-infected BEAS-2B cells (V-RNA-1, 300 ng; V-RNA-2, 900 ng)) for 24 h. ( f ) BEAS-2B cells were infected without (−) or with H1N1 at MOI of 1 and then incubated with DMSO, piceatannol (Piceat, 15 μM), BAY11-7082 (BAY, 10 μM), SC514 (8 μM), or Amlexanol (Amlex, 10 μM) for 24 h. ( g ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con), human p65, IKKβ siRNA-1 (β-1) or IKKβ siRNA-2 (β-2), grown for 72 h, then infected with H1N1 at MOI of 1 for 24 h. ( h ) Primary signal pathway for Runx3 induction by IAV H1N1. ( i ) BEAS-2B cells were treated with control PBS, IFNα (300 U/ml), IFNγ (200 ng/ml), or IFNλ (200 ng/ml) for 24 h. Equal amounts of cell lysates from ( a–i ) were subjected to Western blot analysis with indicated antibodies. Results represent Western blots of three independent experiments. Relative changes in protein levels of Runx3 of three independent experiments were measured by densitometric analysis using ImageJ 1.47 software and normalized to actin and are represented as percentage of control siRNA (n = 3).

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: IAV-induced expression of Runx3 is primarily mediated by MDA5−NF-κB pathway. ( a , b ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con), human MDA5 siRNA-2 (M), TLR3 siRNA-2 (T), both MDA5 and TLR3 siRNAs (M + T), RIG-1 siRNA-1 (S1), or RIG-1 siRNA-2 (S2), grown for 72 h then infected without (−) or with (+) H1N1 PR/8/34 at MOI of 1 for 24 h. ( c,d ) BEAS-2B cells were transfected with control H 2 O (Con), or different amounts of RNA isolated from uninfected BEAS-2B cells (C-RNA) or H1N1-infected BEAS-2B cells (V-RNA) treated with or without calf intestine alkaline phosphatase (CIAP). After 24 h, IFNβ released into the medium was measured by ELISA ( c ) or equal amounts of cell lysates were subjected to Western blot analysis ( d ). ( e )BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con) or human MDA5 siRNA-2 (M), grown for 72 h, then transfected with control H 2 O (Control) or total RNA isolated from H1N1-infected BEAS-2B cells (V-RNA-1, 300 ng; V-RNA-2, 900 ng)) for 24 h. ( f ) BEAS-2B cells were infected without (−) or with H1N1 at MOI of 1 and then incubated with DMSO, piceatannol (Piceat, 15 μM), BAY11-7082 (BAY, 10 μM), SC514 (8 μM), or Amlexanol (Amlex, 10 μM) for 24 h. ( g ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con), human p65, IKKβ siRNA-1 (β-1) or IKKβ siRNA-2 (β-2), grown for 72 h, then infected with H1N1 at MOI of 1 for 24 h. ( h ) Primary signal pathway for Runx3 induction by IAV H1N1. ( i ) BEAS-2B cells were treated with control PBS, IFNα (300 U/ml), IFNγ (200 ng/ml), or IFNλ (200 ng/ml) for 24 h. Equal amounts of cell lysates from ( a–i ) were subjected to Western blot analysis with indicated antibodies. Results represent Western blots of three independent experiments. Relative changes in protein levels of Runx3 of three independent experiments were measured by densitometric analysis using ImageJ 1.47 software and normalized to actin and are represented as percentage of control siRNA (n = 3).

    Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.

    Techniques: Expressing, Transfection, Infection, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Software

    Runx3 induction by poly(I:C) is primarily mediated by MDA5/TLR3−NF-κB pathway. ( a–c ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con or control), human MDA5 siRNA-1 (S1), MDA5 siRNA-2 (S2), MAVS siRNA, TLR3 siRNA-1 (S1), TLR3 siRNA-2 (S2), RIG-1 siRNA-1 (RIG-1-S1), or RIG-1 siRNA-2 (RIG-1-S2), grown for 72 h then treated with control PBS (-), high molecular weight poly(I:C) (2 μg/ml) ( a-b ), or high (H) or low (L) molecular weight poly(I:C) (2 μg/ml) ( c ) for 20 h. ( d ) BEAS-2B cells were incubated with or without poly(I:C) (PIC, 2 μg/ml) in the presence of DMSO, piceatannol (Piceata, 15 μM), BAY11-7082 (BAY, 10 μM), GFX (500 nM), SC514 (8 μM), or Amlexanol (Amlex, 10 μM) for 21 h. ( e–g ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con or Control), human IKKβ siRNA-1 (β-1), IKKβ siRNA-2 (β-2), IKKα siRNA-1 (α-1), IKKα siRNA-2 (α-2), p65 or Stat1 siRNAs, grown for 60 h ( e ) or 72 h ( f,g ), then treated with control PBS (-) or high molecular weight poly(I:C) (2 μg/ml) for 20 h. Equal amounts of cell lysates from ( a-g ) were subjected to Western blot analysis with indicated antibodies. Results represent Western blots of three independent experiments. Relative changes in protein levels of Runx3, MDA5, TLR3, IKKβ or p65 were measured by densitometric analysis using ImageJ 1.47 software, normalized to actin and are represented as percentage of control siRNA (n = 3). ( h ) Signal pathway involved in Runx3 induction by poly(I:C).

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: Runx3 induction by poly(I:C) is primarily mediated by MDA5/TLR3−NF-κB pathway. ( a–c ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con or control), human MDA5 siRNA-1 (S1), MDA5 siRNA-2 (S2), MAVS siRNA, TLR3 siRNA-1 (S1), TLR3 siRNA-2 (S2), RIG-1 siRNA-1 (RIG-1-S1), or RIG-1 siRNA-2 (RIG-1-S2), grown for 72 h then treated with control PBS (-), high molecular weight poly(I:C) (2 μg/ml) ( a-b ), or high (H) or low (L) molecular weight poly(I:C) (2 μg/ml) ( c ) for 20 h. ( d ) BEAS-2B cells were incubated with or without poly(I:C) (PIC, 2 μg/ml) in the presence of DMSO, piceatannol (Piceata, 15 μM), BAY11-7082 (BAY, 10 μM), GFX (500 nM), SC514 (8 μM), or Amlexanol (Amlex, 10 μM) for 21 h. ( e–g ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con or Control), human IKKβ siRNA-1 (β-1), IKKβ siRNA-2 (β-2), IKKα siRNA-1 (α-1), IKKα siRNA-2 (α-2), p65 or Stat1 siRNAs, grown for 60 h ( e ) or 72 h ( f,g ), then treated with control PBS (-) or high molecular weight poly(I:C) (2 μg/ml) for 20 h. Equal amounts of cell lysates from ( a-g ) were subjected to Western blot analysis with indicated antibodies. Results represent Western blots of three independent experiments. Relative changes in protein levels of Runx3, MDA5, TLR3, IKKβ or p65 were measured by densitometric analysis using ImageJ 1.47 software, normalized to actin and are represented as percentage of control siRNA (n = 3). ( h ) Signal pathway involved in Runx3 induction by poly(I:C).

    Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.

    Techniques: Transfection, Molecular Weight, Incubation, Western Blot, Software

    Runx3 is critical for airway epithelial cell apoptosis induced by IAV infection. ( a ) BEAS-2B cells were infected with H1N1 PR/8/34 at MOI of 3 for 24 h in the presence or absence of Z-VAD-FMK (Z-VAD, 20 μM) or necrostatin-1 (Nec-1, 50 μM). Detached and adherent cells were collected separately and counted with trypan blue by using a TC20 automated cell counter and cell death rate (detached dead cells over total detached and adherent cells) was shown. Data are means ± S.E. (n = 3). ( b ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone (Vec) or Runx3, grown for 60 h, then infected with (+) IAV H1N1 at MOI of 1 or treated with control PBS (Control) for 24 h. Representative images (final magnification: ×200) of TUNEL staining and cell apoptotic rate (%) from three independent experiments are shown. Apoptotic rate was determined as TUNEL-positive cells (green color) over total DAPI-stained nuclei by using ImageJ 1.47 software. Data are means ± S.E. (n = 3). **p

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: Runx3 is critical for airway epithelial cell apoptosis induced by IAV infection. ( a ) BEAS-2B cells were infected with H1N1 PR/8/34 at MOI of 3 for 24 h in the presence or absence of Z-VAD-FMK (Z-VAD, 20 μM) or necrostatin-1 (Nec-1, 50 μM). Detached and adherent cells were collected separately and counted with trypan blue by using a TC20 automated cell counter and cell death rate (detached dead cells over total detached and adherent cells) was shown. Data are means ± S.E. (n = 3). ( b ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone (Vec) or Runx3, grown for 60 h, then infected with (+) IAV H1N1 at MOI of 1 or treated with control PBS (Control) for 24 h. Representative images (final magnification: ×200) of TUNEL staining and cell apoptotic rate (%) from three independent experiments are shown. Apoptotic rate was determined as TUNEL-positive cells (green color) over total DAPI-stained nuclei by using ImageJ 1.47 software. Data are means ± S.E. (n = 3). **p

    Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.

    Techniques: Infection, Recombinant, Plasmid Preparation, TUNEL Assay, Staining, Software

    Runx3 is induced by IAV infection in airway epithelial cells. ( a ) BEAS-2B cells were treated with control PBS (mock) or infected with active H1N1 PR/8/34 strain at MOI of 1 or inactivated H1N1 viruses (UV light exposure or incubation in 65 o C for 30 min), grown for 24 h, and cell lysates at equal protein amounts were subjected to Western blotting with Runx3 or NP antibodies. ( b ) BEAS-2B cells were infected without (–) or with ( + ) H1N1 PR/8/34 at various MOIs of 0.05 to 3 for 24 h, and cell lysates at equal protein amounts were subjected to Western blotting with indicated antibodies. ( c,d ) BEAS-2B cells were infected with H1N1 (MOI = 1) for 0–24 h, and the kinetics of Runx3 mRNA and protein expression were determined by quantitative real-time PCR ( c ) and Western blot ( d ), respectively. ( e,f ) BEAS-2B cells ( e ) and SAECs ( f ) were infected without (–) or with ( + ) H3N2 (x:31) A/Aichi/68 or H1N1 PR/8/34 strains at different MOIs for 24 h, and cell lysates at equal protein amounts were subjected to Western blotting with indicated antibodies. ( g ) BEAS-2B cells were infected with H1N1 at various MOIs of 0.5 to 3 in the presence of control DMSO or cycloheximide (CHX, 5 μM) for 16 h. Runx3 mRNA level was measured by RT-PCR and normalized to GAPDH, and relative changes (fold) are shown. Data are means ± S.E. (n = 3). *p

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: Runx3 is induced by IAV infection in airway epithelial cells. ( a ) BEAS-2B cells were treated with control PBS (mock) or infected with active H1N1 PR/8/34 strain at MOI of 1 or inactivated H1N1 viruses (UV light exposure or incubation in 65 o C for 30 min), grown for 24 h, and cell lysates at equal protein amounts were subjected to Western blotting with Runx3 or NP antibodies. ( b ) BEAS-2B cells were infected without (–) or with ( + ) H1N1 PR/8/34 at various MOIs of 0.05 to 3 for 24 h, and cell lysates at equal protein amounts were subjected to Western blotting with indicated antibodies. ( c,d ) BEAS-2B cells were infected with H1N1 (MOI = 1) for 0–24 h, and the kinetics of Runx3 mRNA and protein expression were determined by quantitative real-time PCR ( c ) and Western blot ( d ), respectively. ( e,f ) BEAS-2B cells ( e ) and SAECs ( f ) were infected without (–) or with ( + ) H3N2 (x:31) A/Aichi/68 or H1N1 PR/8/34 strains at different MOIs for 24 h, and cell lysates at equal protein amounts were subjected to Western blotting with indicated antibodies. ( g ) BEAS-2B cells were infected with H1N1 at various MOIs of 0.5 to 3 in the presence of control DMSO or cycloheximide (CHX, 5 μM) for 16 h. Runx3 mRNA level was measured by RT-PCR and normalized to GAPDH, and relative changes (fold) are shown. Data are means ± S.E. (n = 3). *p

    Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.

    Techniques: Infection, Incubation, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Runx3 promotes airway epithelial cell apoptosis induced by IAV viral RNA and dsRNA poly(I:C). ( a–d ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone, Runx3 or PKD3, and grown for 60 h. ( a ) The cells were then transfected with control H 2 O (control), or total RNA isolated from uninfected BEAS-2B cells (C-RNA, 0.5 μg) or H1N1-infected BEAS-2B cells (V-RNA, 0.5 μg) for 24 h. The cellular death rate (detached dead cells over total detached and adherent cells) was determined by using a TC20 automated cell counter with trypan blue. ( b ) The cells were then left untreated or treated 4 h with high molecular weight poly(I:C) (2 μg/ml) in the presence or absence of Z-VAD-FMK (Z-VAD, 20 μM) and the death rate of the cells was determined. ( c ) The cells were treated with control PBS (Control) or poly(I:C) (2 μg/ml) for 4 h and cellular apoptosis was assessed by TUNEL assay. Cell apoptotic rate was determined as TUNEL-positive cells over total DAPI-stained nuclei ( supplementary Fig. S5 ) by using ImageJ 1.47 software. All Data in ( a–c ) are means ± S.E. of three independent experiments. **p

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: Runx3 promotes airway epithelial cell apoptosis induced by IAV viral RNA and dsRNA poly(I:C). ( a–d ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone, Runx3 or PKD3, and grown for 60 h. ( a ) The cells were then transfected with control H 2 O (control), or total RNA isolated from uninfected BEAS-2B cells (C-RNA, 0.5 μg) or H1N1-infected BEAS-2B cells (V-RNA, 0.5 μg) for 24 h. The cellular death rate (detached dead cells over total detached and adherent cells) was determined by using a TC20 automated cell counter with trypan blue. ( b ) The cells were then left untreated or treated 4 h with high molecular weight poly(I:C) (2 μg/ml) in the presence or absence of Z-VAD-FMK (Z-VAD, 20 μM) and the death rate of the cells was determined. ( c ) The cells were treated with control PBS (Control) or poly(I:C) (2 μg/ml) for 4 h and cellular apoptosis was assessed by TUNEL assay. Cell apoptotic rate was determined as TUNEL-positive cells over total DAPI-stained nuclei ( supplementary Fig. S5 ) by using ImageJ 1.47 software. All Data in ( a–c ) are means ± S.E. of three independent experiments. **p

    Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.

    Techniques: Infection, Recombinant, Plasmid Preparation, Transfection, Isolation, Molecular Weight, TUNEL Assay, Staining, Software

    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Journal: Cell Research

    Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    Analysis of TLR4, CD14 and MD2 expression in enriched and L-LME-purified astrocytes. a Western blots show comparable levels of TLR4, CD14 and MD2 in both astrocyte populations. Left panel : representative immunoblot. Right panel : Mean values from duplicate samples. b Confocal microscopy shows co-expression of GFAP ( green ) and TLR4 ( red ) in enriched (L-LME - ) as well as in purified (L-LME + ) astrocytes. Lower frame in each case illustrates GFAP/TLR4 overlap. Nuclei are labelled with DAPI ( blue ). c LPS conjugated with Alexa Fluor® 594 ( red ) co-localizes with GFAP ( green ) in L-LME-purified astrocytes after 30 min of treatment ( arrow ). Nuclei are labelled with DAPI

    Journal: Journal of Neuroinflammation

    Article Title: Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes

    doi: 10.1186/s12974-015-0458-6

    Figure Lengend Snippet: Analysis of TLR4, CD14 and MD2 expression in enriched and L-LME-purified astrocytes. a Western blots show comparable levels of TLR4, CD14 and MD2 in both astrocyte populations. Left panel : representative immunoblot. Right panel : Mean values from duplicate samples. b Confocal microscopy shows co-expression of GFAP ( green ) and TLR4 ( red ) in enriched (L-LME - ) as well as in purified (L-LME + ) astrocytes. Lower frame in each case illustrates GFAP/TLR4 overlap. Nuclei are labelled with DAPI ( blue ). c LPS conjugated with Alexa Fluor® 594 ( red ) co-localizes with GFAP ( green ) in L-LME-purified astrocytes after 30 min of treatment ( arrow ). Nuclei are labelled with DAPI

    Article Snippet: Purified microglia and enriched astrocytes were immunophenotypically characterized by FCM using the following primary antibodies against rat markers: Alexa Fluor® 647 mouse anti-glial fibrillary acidic protein (GFAP) (Cell Signaling Technology Europe, Leiden, The Netherlands); rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) (Wako, Richmond, VA, USA); rabbit anti-TLR2 (sc-10739) polyclonal antibody raised against amino acids 180–354 of TLR2 of human origin (Santa Cruz Biotechnology, Heidelberg, Germany); rabbit anti-rat TLR3 (sc-28999) polyclonal antibody raised against amino acids 26–325 mapping within an N-terminal extracellular domain of TLR4 of mouse origin (Santa Cruz); rabbit anti-rat TLR4 (sc-30002) polyclonal antibody raised against amino acids 339–638 mapping within an N-terminal extracellular domain of TLR4 of mouse origin (Santa Cruz); AlexaFluor® 647 mouse monoclonal antibody IgG1 isotype control (Cell Signaling); AlexaFluor®488 anti-rabbit or anti-mouse secondary antibodies (II Ab) (Life Technologies).

    Techniques: Expressing, Purification, Western Blot, Confocal Microscopy

    MT decreases the expression of TLR-4, MyD88 and caspase-3 proteins in LPS-stimulated BV2 cells. BV2 microglia cells were stimulated with 1 µg with 1 µg/ml LPS for 30 min, followed by treatment with 7.5 µg/ml MT for 24 h prior to

    Journal: Experimental and Therapeutic Medicine

    Article Title: Neuroprotective effect of heat shock protein 60 on matrine-suppressed microglial activation

    doi: 10.3892/etm.2017.4691

    Figure Lengend Snippet: MT decreases the expression of TLR-4, MyD88 and caspase-3 proteins in LPS-stimulated BV2 cells. BV2 microglia cells were stimulated with 1 µg with 1 µg/ml LPS for 30 min, followed by treatment with 7.5 µg/ml MT for 24 h prior to

    Article Snippet: Antibodies directed against caspase-3 (cat. no. 9665), MyD88 (cat. no. 4283) and TLR-4 (cat. no. 2219) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Expressing

    MT decreases the expression of TLR-4, MyD88 and caspase-3 proteins in LPS-stimulated BV2 cells. BV2 microglia cells were stimulated with 1 µg with 1 µg/ml LPS for 30 min, followed by treatment with 7.5 µg/ml MT for 24 h prior to

    Journal: Experimental and Therapeutic Medicine

    Article Title: Neuroprotective effect of heat shock protein 60 on matrine-suppressed microglial activation

    doi: 10.3892/etm.2017.4691

    Figure Lengend Snippet: MT decreases the expression of TLR-4, MyD88 and caspase-3 proteins in LPS-stimulated BV2 cells. BV2 microglia cells were stimulated with 1 µg with 1 µg/ml LPS for 30 min, followed by treatment with 7.5 µg/ml MT for 24 h prior to

    Article Snippet: Antibodies directed against caspase-3 (cat. no. 9665), MyD88 (cat. no. 4283) and TLR-4 (cat. no. 2219) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Expressing

    Angiogenin, Angiopoietin-1, TLR4, NFkB and RAGE expression in the ischemic brain

    Journal: Neuroscience

    Article Title: Neamine induces neuroprotection after acute ischemic stroke in type one diabetic rats

    doi: 10.1016/j.neuroscience.2013.10.071

    Figure Lengend Snippet: Angiogenin, Angiopoietin-1, TLR4, NFkB and RAGE expression in the ischemic brain

    Article Snippet: Antibody against cleaved caspase-3 (rabbit polyclonal IgG; dilution 1:200, Cell Signaling Technology, Danvers, Massachusetts), advanced glycation end products (RAGE, 1:400; Dako, Carpenteria, CA, USA), toll-like receptor 4 (TLR-4) (goat polyclonal IgG; dilution 1:100; Cruz Biotech Inc, Santa Cruz, California), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) (1:500; Abcam, Cambribridge, MA, USA), Angiogenin (monoclonal, 1:500, Abcam Cambridge, MA) and Angiopoietin-1 (Ang1) (Ang1, 1:2,000, Abcam) immunostaining were performed.

    Techniques: Expressing

    Investigation of leptin receptor expression and signalling pathways leading to MMP-1 synthesis in HGFs. Flow cytometry for cell surface leptin receptor expression on unstimulated HGFs (A). Data are representative of similar data from 4 HGF donors. Inset (A): long LEPR isoform (Ob-Rb) and β2m gene expression as assessed by RT-PCR in unstimulated HGFs. Data are representative of similar results from 7 HGF donors. HGFs were stimulated with leptin (10 μg/ml), IL-1 (0.05 ng/ml), pam2CSK4 (50 ng/ml), OSM (5 ng/ml), leptin+IL-1 or leptin+pam2CSK4 for 20 min and lysates immunoblotted with the indicated antibodies including GAPDH as a loading control (B). The blots presented are all derived from the same donor and are representative of similar results from 3 donors stimulated in independent experiments. (C) HGFs were pre-treated with the ERK inhibitor U0126 (7.5 μM), (D) the JNK inhibitor SP600126 (10 μM), (E) STAT3 inhibitor VI (100 μM), or (F) the p38 inhibitor SB203580 (10 μM) for 30 min and then stimulated with leptin (10 μg/ml), IL-1α (0.05 ng/ml), pam2CSK4 (50 ng/ml), leptin+IL-1α or leptin+pam2CSK4 for 24 h. MMP-1 gene expression was assessed by real-time RT-PCR. Data (fold unstimulated DMSO-pre-treated control) are expressed relative to RNAP. Data are presented as mean+SEM of three donors stimulated in independent experiments (n = 4 for each donor). * P

    Journal: PLoS ONE

    Article Title: Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts

    doi: 10.1371/journal.pone.0148024

    Figure Lengend Snippet: Investigation of leptin receptor expression and signalling pathways leading to MMP-1 synthesis in HGFs. Flow cytometry for cell surface leptin receptor expression on unstimulated HGFs (A). Data are representative of similar data from 4 HGF donors. Inset (A): long LEPR isoform (Ob-Rb) and β2m gene expression as assessed by RT-PCR in unstimulated HGFs. Data are representative of similar results from 7 HGF donors. HGFs were stimulated with leptin (10 μg/ml), IL-1 (0.05 ng/ml), pam2CSK4 (50 ng/ml), OSM (5 ng/ml), leptin+IL-1 or leptin+pam2CSK4 for 20 min and lysates immunoblotted with the indicated antibodies including GAPDH as a loading control (B). The blots presented are all derived from the same donor and are representative of similar results from 3 donors stimulated in independent experiments. (C) HGFs were pre-treated with the ERK inhibitor U0126 (7.5 μM), (D) the JNK inhibitor SP600126 (10 μM), (E) STAT3 inhibitor VI (100 μM), or (F) the p38 inhibitor SB203580 (10 μM) for 30 min and then stimulated with leptin (10 μg/ml), IL-1α (0.05 ng/ml), pam2CSK4 (50 ng/ml), leptin+IL-1α or leptin+pam2CSK4 for 24 h. MMP-1 gene expression was assessed by real-time RT-PCR. Data (fold unstimulated DMSO-pre-treated control) are expressed relative to RNAP. Data are presented as mean+SEM of three donors stimulated in independent experiments (n = 4 for each donor). * P

    Article Snippet: Monoclonal antibodies (Abs) against phospho-STAT1 (Y701), phospho-STAT3 (Y705), STAT-3, phospho-p38 (T180/Y182), phospho-JNK (T183/Y185), phospho-ERK1/2 (T202/Y204), ERK1/2 and phospho-Akt (S473) were from Cell-Signalling Technologies (New England Biolabs, Hitchin, UK), GAPDH Ab from Merck-Millipore, HRP-conjugated anti-mouse and anti-rabbit Ig Abs from Dako (Ely, UK), IgG2B mouse LEPR and isotype control PE-conjugated Abs from R & D Systems, IgG2A mouse Toll-like receptor (TLR)2 and TLR4 APC-conjugated Abs were from EBioscience (Hatfield, UK), and an isotype control APC-conjugated Ab was from AbD Serotec (Kidlington, UK).

    Techniques: Expressing, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR

    Angiogenin, Angiopoietin-1, TLR4, NFkB and RAGE expression in the ischemic brain

    Journal: Neuroscience

    Article Title: Neamine induces neuroprotection after acute ischemic stroke in type one diabetic rats

    doi: 10.1016/j.neuroscience.2013.10.071

    Figure Lengend Snippet: Angiogenin, Angiopoietin-1, TLR4, NFkB and RAGE expression in the ischemic brain

    Article Snippet: Antibody against cleaved caspase-3 (rabbit polyclonal IgG; dilution 1:200, Cell Signaling Technology, Danvers, Massachusetts), advanced glycation end products (RAGE, 1:400; Dako, Carpenteria, CA, USA), toll-like receptor 4 (TLR-4) (goat polyclonal IgG; dilution 1:100; Cruz Biotech Inc, Santa Cruz, California), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) (1:500; Abcam, Cambribridge, MA, USA), Angiogenin (monoclonal, 1:500, Abcam Cambridge, MA) and Angiopoietin-1 (Ang1) (Ang1, 1:2,000, Abcam) immunostaining were performed.

    Techniques: Expressing