igg isotype antibody Search Results


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  • 97
    Thermo Fisher isotype specific monoclonal secondary antibodies igg1
    PSD enrichment and interaction of endogenous NPRAP with N-cadherin and the scaffolding proteins ABP, GRIP, and PSD-95 in brain and synaptosomal fractions. A , B , Triton X-100 lysates prepared from cultured cortical neurons, 28–35 DIV, were analyzed by immunoprecipitation. A , Endogenous N-cadherin, but not the NMDAR subunit NR1, coimmunoprecipitated endogenous NPRAP, as determined by Western blotting. This demonstrates that endogenous NPRAP interacts specifically with N-cadherin in neurons. B , The interaction of NPRAP with scaffolding proteins was assayed by immunoprecipitation of ABP, GRIP, and PSD-95 from neuron lysates and Western blotting for NPRAP. i , Immunoprecipitates of cultured cortical neuron lysates (400–500 μg) using antibodies to GRIP and ABP and with <t>IgG</t> (control) were analyzed with anti-NPRAP antibody. NPRAP coimmunoprecipitated with GRIP and ABP, demonstrating interaction in cultured neurons. The original lysate (20 μg) was analyzed in parallel. ii , Immunoprecipitates of cultured cortical neuron lysates (400–500 μg) with antibodies to GRIP, ABP, and synaptophysin (S'physin; a control) were analyzed in parallel with the original lysate (20 μg) by Western blotting. NPRAP coimmunoprecipitated with GRIP and PSD-95, but not with synaptophysin, demonstrating specific interactions. C , NPRAP, N-cadherin, GRIP, and GluR2 are enriched in the PSD. Whole-brain (WB) lysate and the synaptosome and PSD fractions were isolated from adult rat brain, and equal quantities of protein (2 μg) of each fraction were analyzed by Western blotting with the indicated antibodies. NPRAP, N-cadherin, GRIP, and GluR2 were enriched in the PSD, whereas synaptophysin, a control, was absent. D , Synaptosomes contain NPRAP in complexes with GRIP and PSD-95. Synaptosomes were isolated from adult rat brain, and Triton X-100 lysates were analyzed by Western blotting, either directly (2 μg) or after immunoprecipitation (1 mg). i , Immunoprecipitation with anti-GRIP antibody but not with a control IgG coimmunoprecipitated NPRAP. ii , Immunoprecipitation with anti-PSD-95 antibody but not with synaptophysin antibody (control) coimmunoprecipitated NPRAP. Blots were reprobed with antibodies to GRIP, PSD-95, and synaptophysin to verify protein presence in the lysates. IB, Immunoblot; IP, immunoprecipitation.
    Isotype Specific Monoclonal Secondary Antibodies Igg1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore isotype control igg2a
    NFIA and NFIB induced IFN-β transcription. MARC-145 cells were transfected with siRNA control (SC) or different concentrations of si-NFIA (A), si-NFIB (B), NFIA-Flag, or NFIB-Flag (C). Forty-eight hours later, the cells were prepared for qRT-PCR and Western blotting. (D) MARC-145 cells were cotransfected with phRL-TK, p340, and siRNA control (SC) or different concentrations of si-NFIA or si-NFIB, and 48 h later the cells were harvested for dual-luciferase assays. (E) MARC-145 cells were transfected with siRNA control (SC) or different concentrations of si-NFIA or si-NFIB, and 48 h later IFN-β mRNA levels were determined by qRT-PCR. (F) MARC-145 cells were cotransfected with phRL-TK, p340, and pcDNA3.1-Flag (NC) or different concentrations of NFIA-Flag or NFIB-Flag, and 48 h later the cells were harvested for dual-luciferase assays. (G) MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or different concentrations of NFIA-Flag or NFIB-Flag, and 48 h later the cellular IFN-β mRNA levels were determined by qRT-PCR. (H) Schematic diagram indicating the location of the putative NFIA/NFIB binding site (red letters) in the Macaca mulatta IFN-β promoter region. (I) EMSA was performed as described in Materials and Methods. Biotin-labeled 35-bp probes which included putative NFIA/NFIB binding sites (underlined) of the Macaca mulatta IFN-β promoter were used. Lane 1 shows labeled probes alone without nuclear extracts (N.E.) from MARC-145 cells, while lane 2 and lane 4 show labeled probes or labeled mutant probes (Mut-probe) with N.E. Competition assays were conducted by adding an excess (200-fold) of unlabeled wild-type or mutant consensus sequence (lanes 3 and 5). (J) ChIP assays. Formaldehyde-cross-linked chromatin was isolated from MARC-145 cells and was sonicated and immunoprecipitated with anti-NFIA antibody, anti-NFIB antibody, or <t>anti-IgG</t> isotype control antibody. Input DNA and immunoprecipitated DNA were purified and analyzed by qRT-PCR and PCR using primers specific for the IFN-β promoter. (K) MARC-145 cells were cotransfected with phRL-TK, the indicated reporter plasmid (pGL4.17, p340, or Mut-p340), pcDNA3.1-Flag (NC), NFIA-Flag, or NFIB-Flag, and 48 h later the cells were harvested for dual-luciferase assays. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the respective control. *, P
    Isotype Control Igg2a, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    SouthernBiotech isotype igg1 igg2 igg3 or igg4
    NFIA and NFIB induced IFN-β transcription. MARC-145 cells were transfected with siRNA control (SC) or different concentrations of si-NFIA (A), si-NFIB (B), NFIA-Flag, or NFIB-Flag (C). Forty-eight hours later, the cells were prepared for qRT-PCR and Western blotting. (D) MARC-145 cells were cotransfected with phRL-TK, p340, and siRNA control (SC) or different concentrations of si-NFIA or si-NFIB, and 48 h later the cells were harvested for dual-luciferase assays. (E) MARC-145 cells were transfected with siRNA control (SC) or different concentrations of si-NFIA or si-NFIB, and 48 h later IFN-β mRNA levels were determined by qRT-PCR. (F) MARC-145 cells were cotransfected with phRL-TK, p340, and pcDNA3.1-Flag (NC) or different concentrations of NFIA-Flag or NFIB-Flag, and 48 h later the cells were harvested for dual-luciferase assays. (G) MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or different concentrations of NFIA-Flag or NFIB-Flag, and 48 h later the cellular IFN-β mRNA levels were determined by qRT-PCR. (H) Schematic diagram indicating the location of the putative NFIA/NFIB binding site (red letters) in the Macaca mulatta IFN-β promoter region. (I) EMSA was performed as described in Materials and Methods. Biotin-labeled 35-bp probes which included putative NFIA/NFIB binding sites (underlined) of the Macaca mulatta IFN-β promoter were used. Lane 1 shows labeled probes alone without nuclear extracts (N.E.) from MARC-145 cells, while lane 2 and lane 4 show labeled probes or labeled mutant probes (Mut-probe) with N.E. Competition assays were conducted by adding an excess (200-fold) of unlabeled wild-type or mutant consensus sequence (lanes 3 and 5). (J) ChIP assays. Formaldehyde-cross-linked chromatin was isolated from MARC-145 cells and was sonicated and immunoprecipitated with anti-NFIA antibody, anti-NFIB antibody, or <t>anti-IgG</t> isotype control antibody. Input DNA and immunoprecipitated DNA were purified and analyzed by qRT-PCR and PCR using primers specific for the IFN-β promoter. (K) MARC-145 cells were cotransfected with phRL-TK, the indicated reporter plasmid (pGL4.17, p340, or Mut-p340), pcDNA3.1-Flag (NC), NFIA-Flag, or NFIB-Flag, and 48 h later the cells were harvested for dual-luciferase assays. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the respective control. *, P
    Isotype Igg1 Igg2 Igg3 Or Igg4, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems mouse igg1 isotype control antibody
    IL-8 production in Caco-2 cells after treatment with TNF-α in the presence of antibodies to TNFR1 or TNFR2 in the basolateral chamber. α- TNFR1 antibody to TNF-α receptor 1 (15 μg/ml), α- TNFR2 antibody to TNF-α receptor 2 (15 μg/ml), <t>IgG</t> non-specific antibody (30 μg/ml). * p
    Mouse Igg1 Isotype Control Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse igg1 isotype control antibody/product/R&D Systems
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    94
    BioLegend isotype controls igg1 igg4
    IL-8 production in Caco-2 cells after treatment with TNF-α in the presence of antibodies to TNFR1 or TNFR2 in the basolateral chamber. α- TNFR1 antibody to TNF-α receptor 1 (15 μg/ml), α- TNFR2 antibody to TNF-α receptor 2 (15 μg/ml), <t>IgG</t> non-specific antibody (30 μg/ml). * p
    Isotype Controls Igg1 Igg4, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend isotype igg3 antibody
    IL-8 production in Caco-2 cells after treatment with TNF-α in the presence of antibodies to TNFR1 or TNFR2 in the basolateral chamber. α- TNFR1 antibody to TNF-α receptor 1 (15 μg/ml), α- TNFR2 antibody to TNF-α receptor 2 (15 μg/ml), <t>IgG</t> non-specific antibody (30 μg/ml). * p
    Isotype Igg3 Antibody, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/isotype igg3 antibody/product/BioLegend
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    89
    Agilent technologies isotype igg1
    IL-8 production in Caco-2 cells after treatment with TNF-α in the presence of antibodies to TNFR1 or TNFR2 in the basolateral chamber. α- TNFR1 antibody to TNF-α receptor 1 (15 μg/ml), α- TNFR2 antibody to TNF-α receptor 2 (15 μg/ml), <t>IgG</t> non-specific antibody (30 μg/ml). * p
    Isotype Igg1, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson anti igg3 isotype
    Lack of Caspase-10 leads to TNF- α production in response to 5FU and FADD-independent apoptosis. ( a ) (Left) Caspase-8 activity was measured in response to 5FU in HCT.shC10 cells. (Right) Double knockdown clones, HCT.shC10.shctrl, HCT.shC10.shC8 and HCT.shC10.shFADD were tested for apoptosis in response to 5FU. Data are plotted as mean±S.E.M. ( n ≥3). ( b ) 5FU-induced apoptosis was measured in HCT.shC10 cells in the presence of TNF- α and TNFR1-blocking antibodies. <t>IgG1</t> and IgG2a antibodies were used as isotype controls, respectively. Data are plotted as mean±S.E.M. (n≥3). ( c ) TNF- α was measured in supernatants of 5FU-treated HCT.shC10 cells. Data are plotted as mean±S.E.M. ( n =3). ( d ) HCT.shC10 cells after 5FU treatment were analysed by western blot for I κ B- α and phospho-I κ B- α . ( e ) HCT.shC10 cells expressing I κ B-SR (Ad.I κ B-SR) were treated with 5FU, after which TNF- α and apoptosis were measured. Ad.EGFP served as control. Data are plotted as mean±S.E.M. ( n ≥3). ( f ) HCT116 cells silenced for both caspase-10 and caspase-8 were treated with 5FU and TNF- α was measured. Data are plotted as mean±S.E.M. ( n ≥5). ( g ) Western blot for cFLIP in HCT116 and HCT.shC10 cells after 5FU treatment. ( h ) EGFP, cFLIP S , cFLIP L , cFLIP p43 and cFLIP D376N (FLIP DN ) were expressed in HCT116 cells, and then the cells treated with 5FU before TNF- α levels were measured. Data are plotted as mean±S.E.M. ( n ≥2). ( i ) HCT.shC10 cells were silenced for cFLIP (Ad.shFLIP). TNF- α in cell culture supernatants was then determined after 5FU stimulation. Data are plotted as mean±S.E.M. ( n =2). ( j ) Either cFLIP L or cFLIP p43 were expressed in HCT.shC8 cells. After 5FU treatment, TNF- α was measured. Data are plotted as mean±S.E.M. (n≥3). ( k ) Precipitates from a caspase-8 IP were probed for cFLIP, FADD and caspase-8. The lysates were from HCT116 cells overexpressing cFLIP L and treated with 5FU. Cells overexpressing EGFP were used as controls. Input controls are shown on the left. ( l ) End point tumour volumes of HCT.shC10 xenografts treated with 5FU are depicted in relation to starting volumes (set to 100). (animal numbers/group: n =6/shctrl, n =3/shC10].
    Anti Igg3 Isotype, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson isotype igg1 antibodies
    Blocking effect of IL12 mAb on the suppressive effect of CpG on α-GalCer-induced AHR, airway inflammation, and cytokine production. As described in Fig. 3 , AHR to methacholine (A), inflammatory cells (B), and cytokines (C, D, and E) in BAL fluid were measured. IL-12 mAb or the <t>IgG2b</t> isotype was intraperitoneally administered immediately before CpG administration. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). * P
    Isotype Igg1 Antibodies, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore igg isotype control
    Histone kills Leishmania . ( A ) Killing of Leishmania by NETs is inhibited by antihistone antibody. Neutrophils treated with PMA and CytD were exposed to 5 μg/mL of <t>anti-H2A</t> histone or <t>IgG</t> isotypic antibodies, and then incubated with promastigotes (1 cell/0.1 parasite ratio) for 2 h at 35 °C. Schneider's complete medium was added to the cultures and live parasites were counted after 2 days incubation at 26 °C. Results of 4 independent experiments are shown as mean + SEM. PMA raw number: 6.6 × 10 5 + 0.8 × 10 5 promastigotes. *, P
    Igg Isotype Control, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore antibody isotypes igg1
    Antibody isotype patterns. Patterns of induction of anti-S <t>IgG</t> isotypes were compared after three immunizations with alum-based or L-pampo-based L-HBsAg formulations. Each formulation contained 0.5 μg of L-HBsAg. L-HBsAg formulated with L-pampo
    Antibody Isotypes Igg1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PSD enrichment and interaction of endogenous NPRAP with N-cadherin and the scaffolding proteins ABP, GRIP, and PSD-95 in brain and synaptosomal fractions. A , B , Triton X-100 lysates prepared from cultured cortical neurons, 28–35 DIV, were analyzed by immunoprecipitation. A , Endogenous N-cadherin, but not the NMDAR subunit NR1, coimmunoprecipitated endogenous NPRAP, as determined by Western blotting. This demonstrates that endogenous NPRAP interacts specifically with N-cadherin in neurons. B , The interaction of NPRAP with scaffolding proteins was assayed by immunoprecipitation of ABP, GRIP, and PSD-95 from neuron lysates and Western blotting for NPRAP. i , Immunoprecipitates of cultured cortical neuron lysates (400–500 μg) using antibodies to GRIP and ABP and with IgG (control) were analyzed with anti-NPRAP antibody. NPRAP coimmunoprecipitated with GRIP and ABP, demonstrating interaction in cultured neurons. The original lysate (20 μg) was analyzed in parallel. ii , Immunoprecipitates of cultured cortical neuron lysates (400–500 μg) with antibodies to GRIP, ABP, and synaptophysin (S'physin; a control) were analyzed in parallel with the original lysate (20 μg) by Western blotting. NPRAP coimmunoprecipitated with GRIP and PSD-95, but not with synaptophysin, demonstrating specific interactions. C , NPRAP, N-cadherin, GRIP, and GluR2 are enriched in the PSD. Whole-brain (WB) lysate and the synaptosome and PSD fractions were isolated from adult rat brain, and equal quantities of protein (2 μg) of each fraction were analyzed by Western blotting with the indicated antibodies. NPRAP, N-cadherin, GRIP, and GluR2 were enriched in the PSD, whereas synaptophysin, a control, was absent. D , Synaptosomes contain NPRAP in complexes with GRIP and PSD-95. Synaptosomes were isolated from adult rat brain, and Triton X-100 lysates were analyzed by Western blotting, either directly (2 μg) or after immunoprecipitation (1 mg). i , Immunoprecipitation with anti-GRIP antibody but not with a control IgG coimmunoprecipitated NPRAP. ii , Immunoprecipitation with anti-PSD-95 antibody but not with synaptophysin antibody (control) coimmunoprecipitated NPRAP. Blots were reprobed with antibodies to GRIP, PSD-95, and synaptophysin to verify protein presence in the lysates. IB, Immunoblot; IP, immunoprecipitation.

    Journal: The Journal of Neuroscience

    Article Title: Synaptic Anchorage of AMPA Receptors by Cadherins through Neural Plakophilin-Related Arm Protein–AMPA Receptor-Binding Protein Complexes

    doi: 10.1523/JNEUROSCI.1395-07.2007

    Figure Lengend Snippet: PSD enrichment and interaction of endogenous NPRAP with N-cadherin and the scaffolding proteins ABP, GRIP, and PSD-95 in brain and synaptosomal fractions. A , B , Triton X-100 lysates prepared from cultured cortical neurons, 28–35 DIV, were analyzed by immunoprecipitation. A , Endogenous N-cadherin, but not the NMDAR subunit NR1, coimmunoprecipitated endogenous NPRAP, as determined by Western blotting. This demonstrates that endogenous NPRAP interacts specifically with N-cadherin in neurons. B , The interaction of NPRAP with scaffolding proteins was assayed by immunoprecipitation of ABP, GRIP, and PSD-95 from neuron lysates and Western blotting for NPRAP. i , Immunoprecipitates of cultured cortical neuron lysates (400–500 μg) using antibodies to GRIP and ABP and with IgG (control) were analyzed with anti-NPRAP antibody. NPRAP coimmunoprecipitated with GRIP and ABP, demonstrating interaction in cultured neurons. The original lysate (20 μg) was analyzed in parallel. ii , Immunoprecipitates of cultured cortical neuron lysates (400–500 μg) with antibodies to GRIP, ABP, and synaptophysin (S'physin; a control) were analyzed in parallel with the original lysate (20 μg) by Western blotting. NPRAP coimmunoprecipitated with GRIP and PSD-95, but not with synaptophysin, demonstrating specific interactions. C , NPRAP, N-cadherin, GRIP, and GluR2 are enriched in the PSD. Whole-brain (WB) lysate and the synaptosome and PSD fractions were isolated from adult rat brain, and equal quantities of protein (2 μg) of each fraction were analyzed by Western blotting with the indicated antibodies. NPRAP, N-cadherin, GRIP, and GluR2 were enriched in the PSD, whereas synaptophysin, a control, was absent. D , Synaptosomes contain NPRAP in complexes with GRIP and PSD-95. Synaptosomes were isolated from adult rat brain, and Triton X-100 lysates were analyzed by Western blotting, either directly (2 μg) or after immunoprecipitation (1 mg). i , Immunoprecipitation with anti-GRIP antibody but not with a control IgG coimmunoprecipitated NPRAP. ii , Immunoprecipitation with anti-PSD-95 antibody but not with synaptophysin antibody (control) coimmunoprecipitated NPRAP. Blots were reprobed with antibodies to GRIP, PSD-95, and synaptophysin to verify protein presence in the lysates. IB, Immunoblot; IP, immunoprecipitation.

    Article Snippet: Isotype-specific monoclonal secondary antibodies IgG1 (Alexa Fluor 568; 1:1000; Invitrogen), IgG2A (Alexa Fluor 488; 1:1000; Invitrogen), and polyclonal FITC-conjugated secondary antibody (1:250; Jackson ImmunoResearch) were added for 1 h at room temperature.

    Techniques: Scaffolding, Cell Culture, Immunoprecipitation, Western Blot, Isolation

    NFIA and NFIB induced IFN-β transcription. MARC-145 cells were transfected with siRNA control (SC) or different concentrations of si-NFIA (A), si-NFIB (B), NFIA-Flag, or NFIB-Flag (C). Forty-eight hours later, the cells were prepared for qRT-PCR and Western blotting. (D) MARC-145 cells were cotransfected with phRL-TK, p340, and siRNA control (SC) or different concentrations of si-NFIA or si-NFIB, and 48 h later the cells were harvested for dual-luciferase assays. (E) MARC-145 cells were transfected with siRNA control (SC) or different concentrations of si-NFIA or si-NFIB, and 48 h later IFN-β mRNA levels were determined by qRT-PCR. (F) MARC-145 cells were cotransfected with phRL-TK, p340, and pcDNA3.1-Flag (NC) or different concentrations of NFIA-Flag or NFIB-Flag, and 48 h later the cells were harvested for dual-luciferase assays. (G) MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or different concentrations of NFIA-Flag or NFIB-Flag, and 48 h later the cellular IFN-β mRNA levels were determined by qRT-PCR. (H) Schematic diagram indicating the location of the putative NFIA/NFIB binding site (red letters) in the Macaca mulatta IFN-β promoter region. (I) EMSA was performed as described in Materials and Methods. Biotin-labeled 35-bp probes which included putative NFIA/NFIB binding sites (underlined) of the Macaca mulatta IFN-β promoter were used. Lane 1 shows labeled probes alone without nuclear extracts (N.E.) from MARC-145 cells, while lane 2 and lane 4 show labeled probes or labeled mutant probes (Mut-probe) with N.E. Competition assays were conducted by adding an excess (200-fold) of unlabeled wild-type or mutant consensus sequence (lanes 3 and 5). (J) ChIP assays. Formaldehyde-cross-linked chromatin was isolated from MARC-145 cells and was sonicated and immunoprecipitated with anti-NFIA antibody, anti-NFIB antibody, or anti-IgG isotype control antibody. Input DNA and immunoprecipitated DNA were purified and analyzed by qRT-PCR and PCR using primers specific for the IFN-β promoter. (K) MARC-145 cells were cotransfected with phRL-TK, the indicated reporter plasmid (pGL4.17, p340, or Mut-p340), pcDNA3.1-Flag (NC), NFIA-Flag, or NFIB-Flag, and 48 h later the cells were harvested for dual-luciferase assays. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the respective control. *, P

    Journal: Journal of Virology

    Article Title: MicroRNA 373 Facilitates the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Its Negative Regulation of Type I Interferon Induction

    doi: 10.1128/JVI.01311-16

    Figure Lengend Snippet: NFIA and NFIB induced IFN-β transcription. MARC-145 cells were transfected with siRNA control (SC) or different concentrations of si-NFIA (A), si-NFIB (B), NFIA-Flag, or NFIB-Flag (C). Forty-eight hours later, the cells were prepared for qRT-PCR and Western blotting. (D) MARC-145 cells were cotransfected with phRL-TK, p340, and siRNA control (SC) or different concentrations of si-NFIA or si-NFIB, and 48 h later the cells were harvested for dual-luciferase assays. (E) MARC-145 cells were transfected with siRNA control (SC) or different concentrations of si-NFIA or si-NFIB, and 48 h later IFN-β mRNA levels were determined by qRT-PCR. (F) MARC-145 cells were cotransfected with phRL-TK, p340, and pcDNA3.1-Flag (NC) or different concentrations of NFIA-Flag or NFIB-Flag, and 48 h later the cells were harvested for dual-luciferase assays. (G) MARC-145 cells were transfected with pcDNA3.1-Flag (NC) or different concentrations of NFIA-Flag or NFIB-Flag, and 48 h later the cellular IFN-β mRNA levels were determined by qRT-PCR. (H) Schematic diagram indicating the location of the putative NFIA/NFIB binding site (red letters) in the Macaca mulatta IFN-β promoter region. (I) EMSA was performed as described in Materials and Methods. Biotin-labeled 35-bp probes which included putative NFIA/NFIB binding sites (underlined) of the Macaca mulatta IFN-β promoter were used. Lane 1 shows labeled probes alone without nuclear extracts (N.E.) from MARC-145 cells, while lane 2 and lane 4 show labeled probes or labeled mutant probes (Mut-probe) with N.E. Competition assays were conducted by adding an excess (200-fold) of unlabeled wild-type or mutant consensus sequence (lanes 3 and 5). (J) ChIP assays. Formaldehyde-cross-linked chromatin was isolated from MARC-145 cells and was sonicated and immunoprecipitated with anti-NFIA antibody, anti-NFIB antibody, or anti-IgG isotype control antibody. Input DNA and immunoprecipitated DNA were purified and analyzed by qRT-PCR and PCR using primers specific for the IFN-β promoter. (K) MARC-145 cells were cotransfected with phRL-TK, the indicated reporter plasmid (pGL4.17, p340, or Mut-p340), pcDNA3.1-Flag (NC), NFIA-Flag, or NFIB-Flag, and 48 h later the cells were harvested for dual-luciferase assays. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the respective control. *, P

    Article Snippet: The supernatants were incubated with an anti-Ago2 monoclonal antibody or isotype control IgG2a at 4°C overnight and then mixed with protein A-agarose (Sigma) for 2 h. The RNA from the immunoprecipitation product was isolated by the RNeasy minikit (Qiagen, Hilden, Germany).

    Techniques: Transfection, Quantitative RT-PCR, Western Blot, Luciferase, Binding Assay, Labeling, Mutagenesis, Sequencing, Chromatin Immunoprecipitation, Isolation, Sonication, Immunoprecipitation, Purification, Polymerase Chain Reaction, Plasmid Preparation

    NFIA, NFIB, IRAK1, IRAK4, IRF1, and IRF9 were the targets of miR-373, and miR-373 inhibited IFN-β production. (A and B) MARC-145 cells were transfected with control mimic (MC), miR-373 mimic (A), control inhibitor (IC), or miR-373 inhibitor (B), and 48 h later the potential target genes were evaluated by qRT-PCR. (C) MARC-145 cells were infected with PRRSV at an MOI of 1 or mock infected, and 24 h later the cells were harvested and measured by qRT-PCR. (D) Schematic presentation of base pairing between the 3′UTR of Macaca mulatta NFIA (or NFIB) and miR-373. 293T cells were cotransfected with pcDNA 6.2-miR-neg, pmiR-373, or pMut-miR-373 along with the Macaca mulatta NFIA or NFIB 3′UTR or mutant 3′UTR luciferase report plasmids. Luciferase activity then was measured at 48 h p.i. For example, NFIA-1 3′UTR (714–720) was the plasmid that included NFIA 3′ UTR fragments from positions 714 to 720. (E) Schematic presentation of base pairing between the 3′UTR of Macaca mulatta IRAK1, IRAK4, IRF1, or IRF9 and miR-373. 293T cells were cotransfected with pcDNA6.2-miR-neg, pmiR-373, or pMut-miR-373 along with Macaca mulatta IRAK1, IRAK4, IRF1, or IRF9 3′UTR or mutant 3′UTR luciferase report plasmids. Luciferase activity was measured at 48 h p.i. (F and G) MARC-145 cells were transfected with miR-373 mi, control mimic (MC), miR-373 inhibitor, or control inhibitor (IC). Forty-eight hours later, the expression levels of NFIA, NFIB, IRAK1, IRAK4, IRF1, and IRF9 were determined by qRT-PCR (F) and Western blotting (G). (H and I) MARC-145 cells, which were transfected with miR-373 mimic (H) or infected with PRRSV (I), were immunoprecipitated with the Ago2-specific monoclonal antibody or the IgG2a isotype control and protein A-agarose. The RNA levels of NFIA, NFIB, IRAK1, IRAK4, IRF1, IRF9, SIRPA, and miR-373 from the immunoprecipitates then were analyzed by qRT-PCR. (J) MARC-145 cells were cotransfected with Macaca mulatta IFN-β promoter luciferase reporter plasmid (p340), phRL-TK, control mimic (MC), miR-373 mimic, control inhibitor (IC), or miR-373 inhibitor for 40 h, and then the cells were transfected with poly(I·C) (10 μg/ml). Eight hours later, the cells were harvested for dual-luciferase assays. (K) MARC-145 cells were transfected with control mimic (MC), miR-373 mimic, control inhibitor (IC), or miR-373 inhibitor for 40 h, and then the cells were transfected with poly(I·C) (10 μg/ml). Eight hours later, the cellular IFN-β mRNA was determined by qRT-PCR. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the indicated control. *, P

    Journal: Journal of Virology

    Article Title: MicroRNA 373 Facilitates the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Its Negative Regulation of Type I Interferon Induction

    doi: 10.1128/JVI.01311-16

    Figure Lengend Snippet: NFIA, NFIB, IRAK1, IRAK4, IRF1, and IRF9 were the targets of miR-373, and miR-373 inhibited IFN-β production. (A and B) MARC-145 cells were transfected with control mimic (MC), miR-373 mimic (A), control inhibitor (IC), or miR-373 inhibitor (B), and 48 h later the potential target genes were evaluated by qRT-PCR. (C) MARC-145 cells were infected with PRRSV at an MOI of 1 or mock infected, and 24 h later the cells were harvested and measured by qRT-PCR. (D) Schematic presentation of base pairing between the 3′UTR of Macaca mulatta NFIA (or NFIB) and miR-373. 293T cells were cotransfected with pcDNA 6.2-miR-neg, pmiR-373, or pMut-miR-373 along with the Macaca mulatta NFIA or NFIB 3′UTR or mutant 3′UTR luciferase report plasmids. Luciferase activity then was measured at 48 h p.i. For example, NFIA-1 3′UTR (714–720) was the plasmid that included NFIA 3′ UTR fragments from positions 714 to 720. (E) Schematic presentation of base pairing between the 3′UTR of Macaca mulatta IRAK1, IRAK4, IRF1, or IRF9 and miR-373. 293T cells were cotransfected with pcDNA6.2-miR-neg, pmiR-373, or pMut-miR-373 along with Macaca mulatta IRAK1, IRAK4, IRF1, or IRF9 3′UTR or mutant 3′UTR luciferase report plasmids. Luciferase activity was measured at 48 h p.i. (F and G) MARC-145 cells were transfected with miR-373 mi, control mimic (MC), miR-373 inhibitor, or control inhibitor (IC). Forty-eight hours later, the expression levels of NFIA, NFIB, IRAK1, IRAK4, IRF1, and IRF9 were determined by qRT-PCR (F) and Western blotting (G). (H and I) MARC-145 cells, which were transfected with miR-373 mimic (H) or infected with PRRSV (I), were immunoprecipitated with the Ago2-specific monoclonal antibody or the IgG2a isotype control and protein A-agarose. The RNA levels of NFIA, NFIB, IRAK1, IRAK4, IRF1, IRF9, SIRPA, and miR-373 from the immunoprecipitates then were analyzed by qRT-PCR. (J) MARC-145 cells were cotransfected with Macaca mulatta IFN-β promoter luciferase reporter plasmid (p340), phRL-TK, control mimic (MC), miR-373 mimic, control inhibitor (IC), or miR-373 inhibitor for 40 h, and then the cells were transfected with poly(I·C) (10 μg/ml). Eight hours later, the cells were harvested for dual-luciferase assays. (K) MARC-145 cells were transfected with control mimic (MC), miR-373 mimic, control inhibitor (IC), or miR-373 inhibitor for 40 h, and then the cells were transfected with poly(I·C) (10 μg/ml). Eight hours later, the cellular IFN-β mRNA was determined by qRT-PCR. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the indicated control. *, P

    Article Snippet: The supernatants were incubated with an anti-Ago2 monoclonal antibody or isotype control IgG2a at 4°C overnight and then mixed with protein A-agarose (Sigma) for 2 h. The RNA from the immunoprecipitation product was isolated by the RNeasy minikit (Qiagen, Hilden, Germany).

    Techniques: Transfection, Quantitative RT-PCR, Infection, Mutagenesis, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation

    PRRSV infection upregulated miR-373 expression in MARC-145 cells. (A) MARC-145 cells were infected with PRRSV at an MOI of 1 for the indicated times, and the levels of miR-373 expression were measured by qRT-PCR. (B) MARC-145 cells were infected with PRRSV at different MOIs for 24 h, and then the levels of miR-373 expression were measured by qRT-PCR. (C) MARC-145 cells were transfected with pGL-miR-373, phRL-TK, or pGL4.17, and 24 h later the cells were infected with PRRSV at different MOIs. Forty-eight hours later, the cells were subjected to dual-luciferase assays. (D) 293T cells were cotransfected with different truncated miR-373 promoter report plasmids and phRL-TK, and 48 h later the cells were harvested for dual-luciferase assays. (E) miR-373 promoter sequence of Macaca mulatta and human harbored one conserved putative GR binding site and three highly conserved putative Sp1 binding site. 293T cells were cotransfected with the indicated report plasmids and phRL-TK, and 48 h later the cells were harvested for dual-luciferase assays. (Left) Schematic representation of mutation constructs of the Macaca mulatta miR-373 promoter. (Right) Results of dual-luciferase assays. (F) MARC-145 cells were cotransfected with phRL-TK, pGL-miR-373, pcDNA-3.1-Flag (NC), Sp1-Flag (800 ng), siRNA control (SC), different concentrations of si-Sp1, or different doses of Mith, and 48 h later the miR-373 promoter activity was analyzed by dual-luciferase assays and the expression levels of pri-miR-373 and miR-373 were detected by qRT-PCR. Additionally, the expression levels of Sp1 were detected by qRT-PCR and Western blotting of the corresponding group. (G) EMSA was performed as described in Materials and Methods. Biotin-labeled 40-bp probes, which included the putative Sp1 binding site (underlined) of the Macaca mulatta miRNA-373 promoter, were used. Lane 1 shows labeled probes alone without nuclear extracts (N.E.) from MARC-145 cells, while lane 2 and lane 4 show labeled wild-type or mutant probes with N.E. Competition assays were conducted by adding an excess (200-fold) of unlabeled wild-type or mutant consensus sequence (lanes 3 and 5). (H) ChIP assays in MARC-145 cells were performed with anti-Sp1 antibody or anti-IgG isotype control antibody. The input DNA and immunoprecipitated DNA then were purified and analyzed by qRT-PCR and PCR using primers specific for the miR-373 promoter. (I) MARC-145 cells were infected with PRRSV at an MOI of 1 or mock infected for 24 h, and the expression levels of Sp1 were determined by qRT-PCR and Western blotting. (J) MARC-145 cells were cotransfected with phRL-TK, pGL-miR-373, pcDNA-3.1-Flag (NC), siRNA control (SC), different concentrations of Sp1-Flag, different concentrations of si-Sp1, or different doses of Mith, and 24 h later the cells were infected with PRRSV at an MOI of 0.1. Forty-eight hours later, miR-373 promoter activity was analyzed by dual-luciferase assays. (K and L) MARC-145 cells were transfected with pcDNA-3.1-Flag (NC), siRNA control (SC), different concentrations of Sp1-Flag, different concentrations of si-Sp1, or different doses of Mith, and 24 h later the cells were infected with PRRSV at an MOI of 0.1. Forty-eight hours later the expression levels of pri-miR-373 (K) and miR-373 (L) were detected by qRT-PCR. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the indicated control. *, P

    Journal: Journal of Virology

    Article Title: MicroRNA 373 Facilitates the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Its Negative Regulation of Type I Interferon Induction

    doi: 10.1128/JVI.01311-16

    Figure Lengend Snippet: PRRSV infection upregulated miR-373 expression in MARC-145 cells. (A) MARC-145 cells were infected with PRRSV at an MOI of 1 for the indicated times, and the levels of miR-373 expression were measured by qRT-PCR. (B) MARC-145 cells were infected with PRRSV at different MOIs for 24 h, and then the levels of miR-373 expression were measured by qRT-PCR. (C) MARC-145 cells were transfected with pGL-miR-373, phRL-TK, or pGL4.17, and 24 h later the cells were infected with PRRSV at different MOIs. Forty-eight hours later, the cells were subjected to dual-luciferase assays. (D) 293T cells were cotransfected with different truncated miR-373 promoter report plasmids and phRL-TK, and 48 h later the cells were harvested for dual-luciferase assays. (E) miR-373 promoter sequence of Macaca mulatta and human harbored one conserved putative GR binding site and three highly conserved putative Sp1 binding site. 293T cells were cotransfected with the indicated report plasmids and phRL-TK, and 48 h later the cells were harvested for dual-luciferase assays. (Left) Schematic representation of mutation constructs of the Macaca mulatta miR-373 promoter. (Right) Results of dual-luciferase assays. (F) MARC-145 cells were cotransfected with phRL-TK, pGL-miR-373, pcDNA-3.1-Flag (NC), Sp1-Flag (800 ng), siRNA control (SC), different concentrations of si-Sp1, or different doses of Mith, and 48 h later the miR-373 promoter activity was analyzed by dual-luciferase assays and the expression levels of pri-miR-373 and miR-373 were detected by qRT-PCR. Additionally, the expression levels of Sp1 were detected by qRT-PCR and Western blotting of the corresponding group. (G) EMSA was performed as described in Materials and Methods. Biotin-labeled 40-bp probes, which included the putative Sp1 binding site (underlined) of the Macaca mulatta miRNA-373 promoter, were used. Lane 1 shows labeled probes alone without nuclear extracts (N.E.) from MARC-145 cells, while lane 2 and lane 4 show labeled wild-type or mutant probes with N.E. Competition assays were conducted by adding an excess (200-fold) of unlabeled wild-type or mutant consensus sequence (lanes 3 and 5). (H) ChIP assays in MARC-145 cells were performed with anti-Sp1 antibody or anti-IgG isotype control antibody. The input DNA and immunoprecipitated DNA then were purified and analyzed by qRT-PCR and PCR using primers specific for the miR-373 promoter. (I) MARC-145 cells were infected with PRRSV at an MOI of 1 or mock infected for 24 h, and the expression levels of Sp1 were determined by qRT-PCR and Western blotting. (J) MARC-145 cells were cotransfected with phRL-TK, pGL-miR-373, pcDNA-3.1-Flag (NC), siRNA control (SC), different concentrations of Sp1-Flag, different concentrations of si-Sp1, or different doses of Mith, and 24 h later the cells were infected with PRRSV at an MOI of 0.1. Forty-eight hours later, miR-373 promoter activity was analyzed by dual-luciferase assays. (K and L) MARC-145 cells were transfected with pcDNA-3.1-Flag (NC), siRNA control (SC), different concentrations of Sp1-Flag, different concentrations of si-Sp1, or different doses of Mith, and 24 h later the cells were infected with PRRSV at an MOI of 0.1. Forty-eight hours later the expression levels of pri-miR-373 (K) and miR-373 (L) were detected by qRT-PCR. Results are expressed as means ± SD from three independent experiments. P values were calculated using Student's t test. An asterisk indicates a comparison with the indicated control. *, P

    Article Snippet: The supernatants were incubated with an anti-Ago2 monoclonal antibody or isotype control IgG2a at 4°C overnight and then mixed with protein A-agarose (Sigma) for 2 h. The RNA from the immunoprecipitation product was isolated by the RNeasy minikit (Qiagen, Hilden, Germany).

    Techniques: Infection, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Sequencing, Binding Assay, Mutagenesis, Construct, Activity Assay, Western Blot, Labeling, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Polymerase Chain Reaction

    Membrane lipid organization and α5 integrin exposure on HeLa cells incubated with microorganisms. (A) HeLa cells were incubated with L. crispatus BC5, S. agalactiae , E. faecalis , or B. subtilis for 1 h and then stained with NR. (B) HeLa cells were incubated with L. crispatus BC5, S. agalactiae , E. faecalis , B. subtilis for 1 h and then stained for α5 integrin subunit. IgG represents specificity staining control. Representative micrographs are shown. Experiments were repeated at least three times with similar results. Bar: 20 μm.

    Journal: Frontiers in Microbiology

    Article Title: Lactobacillus crispatus BC5 Interferes With Chlamydia trachomatis Infectivity Through Integrin Modulation in Cervical Cells

    doi: 10.3389/fmicb.2018.02630

    Figure Lengend Snippet: Membrane lipid organization and α5 integrin exposure on HeLa cells incubated with microorganisms. (A) HeLa cells were incubated with L. crispatus BC5, S. agalactiae , E. faecalis , or B. subtilis for 1 h and then stained with NR. (B) HeLa cells were incubated with L. crispatus BC5, S. agalactiae , E. faecalis , B. subtilis for 1 h and then stained for α5 integrin subunit. IgG represents specificity staining control. Representative micrographs are shown. Experiments were repeated at least three times with similar results. Bar: 20 μm.

    Article Snippet: As a specificity control for α5 integrin subunit signal, untreated Hela cells were also stained with IgG isotype (1:500 in 1% BSA/PBS, Sigma-Aldrich), followed by Alexa 568-conjugated secondary antibody.

    Techniques: Incubation, Staining

    Inhibition of C. trachomatis infection by α5 integrin subunit blocking or ITGA5 gene silencing. HeLa cells were treated or not with an anti-CD49e antibody or control IgG for 1 h, then infected with CT EBs for 48 h. (A,B) Specimens were stained for chlamydial membrane lipopolysaccharide antigen. Representative micrographs are shown. C. trachomatis infectivity was evaluated as number of IFUs/microscopic field. Results were expressed in percentage compared with control, taken as 100%. Bars represent median values, error bars represent median absolute deviations. Statistical significance was calculated vs. control. ∗ P ≤ 0.01. (C,D) Western blotting of α5 integrin subunit expression in control, ITGA 5 siRNA and scramble Hela cells, evaluated at 48 and 120 h post-siRNA. Quantification of α5 integrin subunit was normalized on β-actin. Bars represent mean values based on three independent experiments, error bars represent standard deviations. (E,F) HeLa cells treated with siRNA or scramble were infected with CT EBs for 48 h, and then stained for chlamydial membrane lipopolysaccharide antigen. Bar: 20 μm. Results were expressed in percentage compared with scramble, taken as 100%. Bars represent median values, error bars represent median absolute deviations. Statistical significance was calculated vs. control. ∗ P ≤ 0.01.

    Journal: Frontiers in Microbiology

    Article Title: Lactobacillus crispatus BC5 Interferes With Chlamydia trachomatis Infectivity Through Integrin Modulation in Cervical Cells

    doi: 10.3389/fmicb.2018.02630

    Figure Lengend Snippet: Inhibition of C. trachomatis infection by α5 integrin subunit blocking or ITGA5 gene silencing. HeLa cells were treated or not with an anti-CD49e antibody or control IgG for 1 h, then infected with CT EBs for 48 h. (A,B) Specimens were stained for chlamydial membrane lipopolysaccharide antigen. Representative micrographs are shown. C. trachomatis infectivity was evaluated as number of IFUs/microscopic field. Results were expressed in percentage compared with control, taken as 100%. Bars represent median values, error bars represent median absolute deviations. Statistical significance was calculated vs. control. ∗ P ≤ 0.01. (C,D) Western blotting of α5 integrin subunit expression in control, ITGA 5 siRNA and scramble Hela cells, evaluated at 48 and 120 h post-siRNA. Quantification of α5 integrin subunit was normalized on β-actin. Bars represent mean values based on three independent experiments, error bars represent standard deviations. (E,F) HeLa cells treated with siRNA or scramble were infected with CT EBs for 48 h, and then stained for chlamydial membrane lipopolysaccharide antigen. Bar: 20 μm. Results were expressed in percentage compared with scramble, taken as 100%. Bars represent median values, error bars represent median absolute deviations. Statistical significance was calculated vs. control. ∗ P ≤ 0.01.

    Article Snippet: As a specificity control for α5 integrin subunit signal, untreated Hela cells were also stained with IgG isotype (1:500 in 1% BSA/PBS, Sigma-Aldrich), followed by Alexa 568-conjugated secondary antibody.

    Techniques: Inhibition, Infection, Blocking Assay, Staining, Western Blot, Expressing

    Protective effects of polyclonal anti-dgA IgG and anti-RTA 31RA aptamer on ricin-inhibited luciferase activity. A: Protective effects of anti-dgA IgG against ricin-inhibited luciferase activity, but not anisomycin-inhibited luciferase activity; B: Protective

    Journal:

    Article Title: Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

    doi: 10.3748/wjg.14.6360

    Figure Lengend Snippet: Protective effects of polyclonal anti-dgA IgG and anti-RTA 31RA aptamer on ricin-inhibited luciferase activity. A: Protective effects of anti-dgA IgG against ricin-inhibited luciferase activity, but not anisomycin-inhibited luciferase activity; B: Protective

    Article Snippet: Isotype control mouse IgG, anisomycin and doxycycline were obtained from Sigma-Aldrich.

    Techniques: Luciferase, Activity Assay

    Protective effects of polyclonal anti-dgA IgG and anti-RTA 31RA aptamer on ricin-induced cell cytotoxicity as measured by MTS assay. A: Protective effects of anti-dgA IgG against ricin-induced cytotoxicity, but not anisomycin-induced cytotoxicity; B:

    Journal:

    Article Title: Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

    doi: 10.3748/wjg.14.6360

    Figure Lengend Snippet: Protective effects of polyclonal anti-dgA IgG and anti-RTA 31RA aptamer on ricin-induced cell cytotoxicity as measured by MTS assay. A: Protective effects of anti-dgA IgG against ricin-induced cytotoxicity, but not anisomycin-induced cytotoxicity; B:

    Article Snippet: Isotype control mouse IgG, anisomycin and doxycycline were obtained from Sigma-Aldrich.

    Techniques: MTS Assay

    Parasite-specific antibodies in sera from Trypanosoma cruzi -infected mice. T. cruzi -specific IgM and IgG isotypes titres were determined by ELISA in sera from normal (day 0) or T. cruzi -infected mice at different days post-infection. Test sera were considered

    Journal: Immunology

    Article Title: Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies

    doi: 10.1111/j.1365-2567.2010.03347.x

    Figure Lengend Snippet: Parasite-specific antibodies in sera from Trypanosoma cruzi -infected mice. T. cruzi -specific IgM and IgG isotypes titres were determined by ELISA in sera from normal (day 0) or T. cruzi -infected mice at different days post-infection. Test sera were considered

    Article Snippet: Total IgM and IgG isotype levels (in ng/ml) were determined by ELISA as previously described., In brief, plates were coated with 2·5 μg/ml of the isotype-specific goat anti-mouse antibody (IgM, IgG1, IgG2a, IgG2b and IgG3; Sigma Aldrich) overnight at 4°, and blocked with 1% BSA.

    Techniques: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Immunoglobulin production by spleen cells from Trypanosoma cruzi -infected mice. IgM and IgG isotype concentrations, determined by ELISA, in culture supernatant of splenic cells obtained from normal (day 0) or T. cruzi -infected mice at different days post-infection.

    Journal: Immunology

    Article Title: Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies

    doi: 10.1111/j.1365-2567.2010.03347.x

    Figure Lengend Snippet: Immunoglobulin production by spleen cells from Trypanosoma cruzi -infected mice. IgM and IgG isotype concentrations, determined by ELISA, in culture supernatant of splenic cells obtained from normal (day 0) or T. cruzi -infected mice at different days post-infection.

    Article Snippet: Total IgM and IgG isotype levels (in ng/ml) were determined by ELISA as previously described., In brief, plates were coated with 2·5 μg/ml of the isotype-specific goat anti-mouse antibody (IgM, IgG1, IgG2a, IgG2b and IgG3; Sigma Aldrich) overnight at 4°, and blocked with 1% BSA.

    Techniques: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    IL-8 production in Caco-2 cells after treatment with TNF-α in the presence of antibodies to TNFR1 or TNFR2 in the basolateral chamber. α- TNFR1 antibody to TNF-α receptor 1 (15 μg/ml), α- TNFR2 antibody to TNF-α receptor 2 (15 μg/ml), IgG non-specific antibody (30 μg/ml). * p

    Journal: Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract

    Article Title: TNF-α Induces Vectorial Secretion of IL-8 in Caco-2 Cells

    doi: 10.1007/s11605-010-1321-9

    Figure Lengend Snippet: IL-8 production in Caco-2 cells after treatment with TNF-α in the presence of antibodies to TNFR1 or TNFR2 in the basolateral chamber. α- TNFR1 antibody to TNF-α receptor 1 (15 μg/ml), α- TNFR2 antibody to TNF-α receptor 2 (15 μg/ml), IgG non-specific antibody (30 μg/ml). * p

    Article Snippet: Recombinant human TNF-α, mouse monoclonal anti-human TNF receptor 1 (TNFR1) antibody, mouse monoclonal anti-human TNF receptor 2 (TNFR2) antibody, mouse IgG1 isotype control antibody and enzyme-linked immunosorbent assay (ELISA) kits were purchased from R & D Systems (Minneapolis, MN, USA).

    Techniques:

    IL-8 production in Caco-2 cells after treatment with TNF-α in the presence of antibodies to TNFR1 or TNFR2 in the apical chamber. α- TNFR1 antibody to TNF-α receptor 1 (15 μg/ml), α- TNFR2 antibody to TNF-α receptor 2 (15 μg/ml), IgG isotype control antibody (30 μg/ml). * p

    Journal: Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract

    Article Title: TNF-α Induces Vectorial Secretion of IL-8 in Caco-2 Cells

    doi: 10.1007/s11605-010-1321-9

    Figure Lengend Snippet: IL-8 production in Caco-2 cells after treatment with TNF-α in the presence of antibodies to TNFR1 or TNFR2 in the apical chamber. α- TNFR1 antibody to TNF-α receptor 1 (15 μg/ml), α- TNFR2 antibody to TNF-α receptor 2 (15 μg/ml), IgG isotype control antibody (30 μg/ml). * p

    Article Snippet: Recombinant human TNF-α, mouse monoclonal anti-human TNF receptor 1 (TNFR1) antibody, mouse monoclonal anti-human TNF receptor 2 (TNFR2) antibody, mouse IgG1 isotype control antibody and enzyme-linked immunosorbent assay (ELISA) kits were purchased from R & D Systems (Minneapolis, MN, USA).

    Techniques:

    (A) PET images of NCI/ADR-RES tumor-bearing mice at 4, 24, and 48 h post injection of 64 Cu-DOTA-Pab-IR800 and 64 Cu-DOTA-IgG. The tumor sites are marked in red circles. (B) The quantitative analysis of tumor, liver, kidney, and muscle uptakes of 64 Cu-DOTA-Pab-IR800 (upper) and 64 Cu-DOTA-IgG (lower) derived from PET images.

    Journal: Molecular pharmaceutics

    Article Title: Molecular Imaging of P-glycoprotein in Chemoresistant Tumors using a Dual-Modality PET/Fluorescence Probe

    doi: 10.1021/acs.molpharmaceut.7b00420

    Figure Lengend Snippet: (A) PET images of NCI/ADR-RES tumor-bearing mice at 4, 24, and 48 h post injection of 64 Cu-DOTA-Pab-IR800 and 64 Cu-DOTA-IgG. The tumor sites are marked in red circles. (B) The quantitative analysis of tumor, liver, kidney, and muscle uptakes of 64 Cu-DOTA-Pab-IR800 (upper) and 64 Cu-DOTA-IgG (lower) derived from PET images.

    Article Snippet: Mouse IgG1 isotype control (IgG) was purchased from R & D Systems, Inc. (Minneapolis, MN, USA).

    Techniques: Positron Emission Tomography, Mouse Assay, Injection, Derivative Assay

    Biodistribution of 64 Cu-DOTA-Pab-IR800 and 64 Cu-DOTA-IgG in mice bearing NCI/ADR-RES xenograft. Mice were injected with 64 Cu-DOTA-Pab-IR800 and 64 Cu-DOTA-IgG, and were sacrificed 48h post injection. Tissues and organs of interest were excised and weighed. Radioactivity was measured and the tracer uptake was calculated as %ID/g. The tumor uptake of 64 Cu-DOTA-Pab-IR800 was significantly higher than that of 64 Cu-DOTA-IgG at 48 h post injection

    Journal: Molecular pharmaceutics

    Article Title: Molecular Imaging of P-glycoprotein in Chemoresistant Tumors using a Dual-Modality PET/Fluorescence Probe

    doi: 10.1021/acs.molpharmaceut.7b00420

    Figure Lengend Snippet: Biodistribution of 64 Cu-DOTA-Pab-IR800 and 64 Cu-DOTA-IgG in mice bearing NCI/ADR-RES xenograft. Mice were injected with 64 Cu-DOTA-Pab-IR800 and 64 Cu-DOTA-IgG, and were sacrificed 48h post injection. Tissues and organs of interest were excised and weighed. Radioactivity was measured and the tracer uptake was calculated as %ID/g. The tumor uptake of 64 Cu-DOTA-Pab-IR800 was significantly higher than that of 64 Cu-DOTA-IgG at 48 h post injection

    Article Snippet: Mouse IgG1 isotype control (IgG) was purchased from R & D Systems, Inc. (Minneapolis, MN, USA).

    Techniques: Mouse Assay, Injection, Radioactivity

    Transmammary transfer of BMP9 and BMP10 blocking Abs leads to abnormal hypervascularization in neonatal retinas. ( A – C ”) Representative images of fluorescent isolectin B4-stained retinas either from P6 neonates fed for 3 days by dams injected on P3 with PBS ( A ), control IgG2a/b Abs ( B ), or BMP9/10 blocking Abs ( C – C ”). ( C’ , C” ) are higher magnification images of the relevant boxed areas in C. a, artery; v, vein; scale bars, 500 μm. ( D–L ) Higher magnification showing retinal vasculature fields between an artery and a vein (Plexus, D–F ), or at the front of an artery ( G–I ) or a vein ( J–L ) from neonates treated as in ( A–C ). Scale bars, 100 μm. ( M–O ) Scatter plots showing the vascular density on retinal ‘petals’ at the plexus ( M ), artery front ( N ), or vein front ( O ). Data represent mean ± s.e.m. (n = 6 pups per group from 2 dams); **** P

    Journal: Scientific Reports

    Article Title: A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10

    doi: 10.1038/srep37366

    Figure Lengend Snippet: Transmammary transfer of BMP9 and BMP10 blocking Abs leads to abnormal hypervascularization in neonatal retinas. ( A – C ”) Representative images of fluorescent isolectin B4-stained retinas either from P6 neonates fed for 3 days by dams injected on P3 with PBS ( A ), control IgG2a/b Abs ( B ), or BMP9/10 blocking Abs ( C – C ”). ( C’ , C” ) are higher magnification images of the relevant boxed areas in C. a, artery; v, vein; scale bars, 500 μm. ( D–L ) Higher magnification showing retinal vasculature fields between an artery and a vein (Plexus, D–F ), or at the front of an artery ( G–I ) or a vein ( J–L ) from neonates treated as in ( A–C ). Scale bars, 100 μm. ( M–O ) Scatter plots showing the vascular density on retinal ‘petals’ at the plexus ( M ), artery front ( N ), or vein front ( O ). Data represent mean ± s.e.m. (n = 6 pups per group from 2 dams); **** P

    Article Snippet: Ab injections and transmammary transfer of Abs via lactation Lactating dams were injected i.p. once on P3 with PBS, mouse monoclonal isotype control Abs (15 mg/kg, IgG2b, MAB004; 15 mg/kg, IgG2a, MAB003; R & D Systems), or mouse monoclonal anti-BMP9 and anti-BMP10 Abs (15 mg/kg, IgG2b, MAB3209; 15 mg/kg, IgG2a, MAB2926; R & D Systems, respectively).

    Techniques: Blocking Assay, Staining, Injection

    Transmammary transfer of BMP9 and BMP10 blocking Abs into the circulation of mouse neonates. ( A–D ) ELISAs were performed to measure IgG2a ( A , C ) and anti-BMP9 Ab ( B , D ) levels in the serum of P6 neonates treated at P3 with vehicle (PBS), isotype control IgGs (IgG2a/2b), or anti-BMP9/10 Abs. Neonates were treated during lactation either from dams injected i.p. with ( A , B ), or by direct i.p. injections of ( C , D ), the different Abs or vehicle. Data represent mean ± s.e.m. (n = 5–7 pups per group from 2 dams); **** P

    Journal: Scientific Reports

    Article Title: A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10

    doi: 10.1038/srep37366

    Figure Lengend Snippet: Transmammary transfer of BMP9 and BMP10 blocking Abs into the circulation of mouse neonates. ( A–D ) ELISAs were performed to measure IgG2a ( A , C ) and anti-BMP9 Ab ( B , D ) levels in the serum of P6 neonates treated at P3 with vehicle (PBS), isotype control IgGs (IgG2a/2b), or anti-BMP9/10 Abs. Neonates were treated during lactation either from dams injected i.p. with ( A , B ), or by direct i.p. injections of ( C , D ), the different Abs or vehicle. Data represent mean ± s.e.m. (n = 5–7 pups per group from 2 dams); **** P

    Article Snippet: Ab injections and transmammary transfer of Abs via lactation Lactating dams were injected i.p. once on P3 with PBS, mouse monoclonal isotype control Abs (15 mg/kg, IgG2b, MAB004; 15 mg/kg, IgG2a, MAB003; R & D Systems), or mouse monoclonal anti-BMP9 and anti-BMP10 Abs (15 mg/kg, IgG2b, MAB3209; 15 mg/kg, IgG2a, MAB2926; R & D Systems, respectively).

    Techniques: Blocking Assay, Injection

    Transmammary transfer of BMP9 and BMP10 blocking Abs induces AVMs in neonatal retinas. ( A,B ) Representative images of blue latex-perfused retinal vasculature of P6 neonates fed for 3 days by dams injected on P3 with control IgG2a/b Abs ( A ) or BMP9/10 blocking Abs ( B ). Arrows in B indicate AVMs. Scale bars, 500 μm. ( C ) Scheme depicting the method employed for the quantification of the number of latex dye-positive vessels. ( D ) Histogram showing the total number of vascular crosses. ( E ) Histogram showing the number of vascular crosses per concentric circle on circles 1 to 3. Data represent mean ± s.e.m. (n = 5–7 pups per group from 2 dams); **** P

    Journal: Scientific Reports

    Article Title: A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10

    doi: 10.1038/srep37366

    Figure Lengend Snippet: Transmammary transfer of BMP9 and BMP10 blocking Abs induces AVMs in neonatal retinas. ( A,B ) Representative images of blue latex-perfused retinal vasculature of P6 neonates fed for 3 days by dams injected on P3 with control IgG2a/b Abs ( A ) or BMP9/10 blocking Abs ( B ). Arrows in B indicate AVMs. Scale bars, 500 μm. ( C ) Scheme depicting the method employed for the quantification of the number of latex dye-positive vessels. ( D ) Histogram showing the total number of vascular crosses. ( E ) Histogram showing the number of vascular crosses per concentric circle on circles 1 to 3. Data represent mean ± s.e.m. (n = 5–7 pups per group from 2 dams); **** P

    Article Snippet: Ab injections and transmammary transfer of Abs via lactation Lactating dams were injected i.p. once on P3 with PBS, mouse monoclonal isotype control Abs (15 mg/kg, IgG2b, MAB004; 15 mg/kg, IgG2a, MAB003; R & D Systems), or mouse monoclonal anti-BMP9 and anti-BMP10 Abs (15 mg/kg, IgG2b, MAB3209; 15 mg/kg, IgG2a, MAB2926; R & D Systems, respectively).

    Techniques: Blocking Assay, Injection

    Gene expression changes in BMP9/10-immunoblocked retinas and ALK1-Fc-treated HUVECs. ( A ) RNA-Seq heat map displaying differently expressed genes in mouse whole retinas following transmammary transfer of anti-BMP9/10 or control IgG2a/2b Abs (n = 6 pups per group from 1 dam). ( B ) HUVECs were treated or not (Ctrl) with ALK1-Fc (1 μg/mL, 24 h). Cell extracts were then analyzed by WB using Abs directed against the indicated proteins. ( C ) Densitometric analyses and quantification of phospho-Smad1/5/8, ID1, and ANG2 relative levels in three independent experiments as in ( B ). ( D ) ECs isolated from retinas of pups fed for 3 days by dams injected on P3 with control IgG2a/b Abs (Ctrl) or BMP9/10 blocking Abs (anti-BMP9/10) were analyzed for Id1 and Angpt2 mRNA levels by RT-qPCR. The results are expressed as relative levels of the control condition (n = 3 determinations, n = 6 pups per group from 1 dam). Data in ( C , D ) are mean ± s.e.m.; *** P

    Journal: Scientific Reports

    Article Title: A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10

    doi: 10.1038/srep37366

    Figure Lengend Snippet: Gene expression changes in BMP9/10-immunoblocked retinas and ALK1-Fc-treated HUVECs. ( A ) RNA-Seq heat map displaying differently expressed genes in mouse whole retinas following transmammary transfer of anti-BMP9/10 or control IgG2a/2b Abs (n = 6 pups per group from 1 dam). ( B ) HUVECs were treated or not (Ctrl) with ALK1-Fc (1 μg/mL, 24 h). Cell extracts were then analyzed by WB using Abs directed against the indicated proteins. ( C ) Densitometric analyses and quantification of phospho-Smad1/5/8, ID1, and ANG2 relative levels in three independent experiments as in ( B ). ( D ) ECs isolated from retinas of pups fed for 3 days by dams injected on P3 with control IgG2a/b Abs (Ctrl) or BMP9/10 blocking Abs (anti-BMP9/10) were analyzed for Id1 and Angpt2 mRNA levels by RT-qPCR. The results are expressed as relative levels of the control condition (n = 3 determinations, n = 6 pups per group from 1 dam). Data in ( C , D ) are mean ± s.e.m.; *** P

    Article Snippet: Ab injections and transmammary transfer of Abs via lactation Lactating dams were injected i.p. once on P3 with PBS, mouse monoclonal isotype control Abs (15 mg/kg, IgG2b, MAB004; 15 mg/kg, IgG2a, MAB003; R & D Systems), or mouse monoclonal anti-BMP9 and anti-BMP10 Abs (15 mg/kg, IgG2b, MAB3209; 15 mg/kg, IgG2a, MAB2926; R & D Systems, respectively).

    Techniques: Expressing, RNA Sequencing Assay, Western Blot, Isolation, Injection, Blocking Assay, Quantitative RT-PCR

    Lack of Caspase-10 leads to TNF- α production in response to 5FU and FADD-independent apoptosis. ( a ) (Left) Caspase-8 activity was measured in response to 5FU in HCT.shC10 cells. (Right) Double knockdown clones, HCT.shC10.shctrl, HCT.shC10.shC8 and HCT.shC10.shFADD were tested for apoptosis in response to 5FU. Data are plotted as mean±S.E.M. ( n ≥3). ( b ) 5FU-induced apoptosis was measured in HCT.shC10 cells in the presence of TNF- α and TNFR1-blocking antibodies. IgG1 and IgG2a antibodies were used as isotype controls, respectively. Data are plotted as mean±S.E.M. (n≥3). ( c ) TNF- α was measured in supernatants of 5FU-treated HCT.shC10 cells. Data are plotted as mean±S.E.M. ( n =3). ( d ) HCT.shC10 cells after 5FU treatment were analysed by western blot for I κ B- α and phospho-I κ B- α . ( e ) HCT.shC10 cells expressing I κ B-SR (Ad.I κ B-SR) were treated with 5FU, after which TNF- α and apoptosis were measured. Ad.EGFP served as control. Data are plotted as mean±S.E.M. ( n ≥3). ( f ) HCT116 cells silenced for both caspase-10 and caspase-8 were treated with 5FU and TNF- α was measured. Data are plotted as mean±S.E.M. ( n ≥5). ( g ) Western blot for cFLIP in HCT116 and HCT.shC10 cells after 5FU treatment. ( h ) EGFP, cFLIP S , cFLIP L , cFLIP p43 and cFLIP D376N (FLIP DN ) were expressed in HCT116 cells, and then the cells treated with 5FU before TNF- α levels were measured. Data are plotted as mean±S.E.M. ( n ≥2). ( i ) HCT.shC10 cells were silenced for cFLIP (Ad.shFLIP). TNF- α in cell culture supernatants was then determined after 5FU stimulation. Data are plotted as mean±S.E.M. ( n =2). ( j ) Either cFLIP L or cFLIP p43 were expressed in HCT.shC8 cells. After 5FU treatment, TNF- α was measured. Data are plotted as mean±S.E.M. (n≥3). ( k ) Precipitates from a caspase-8 IP were probed for cFLIP, FADD and caspase-8. The lysates were from HCT116 cells overexpressing cFLIP L and treated with 5FU. Cells overexpressing EGFP were used as controls. Input controls are shown on the left. ( l ) End point tumour volumes of HCT.shC10 xenografts treated with 5FU are depicted in relation to starting volumes (set to 100). (animal numbers/group: n =6/shctrl, n =3/shC10].

    Journal: Cell Death and Differentiation

    Article Title: Caspase-10: a molecular switch from cell-autonomous apoptosis to communal cell death in response to chemotherapeutic drug treatment

    doi: 10.1038/cdd.2017.164

    Figure Lengend Snippet: Lack of Caspase-10 leads to TNF- α production in response to 5FU and FADD-independent apoptosis. ( a ) (Left) Caspase-8 activity was measured in response to 5FU in HCT.shC10 cells. (Right) Double knockdown clones, HCT.shC10.shctrl, HCT.shC10.shC8 and HCT.shC10.shFADD were tested for apoptosis in response to 5FU. Data are plotted as mean±S.E.M. ( n ≥3). ( b ) 5FU-induced apoptosis was measured in HCT.shC10 cells in the presence of TNF- α and TNFR1-blocking antibodies. IgG1 and IgG2a antibodies were used as isotype controls, respectively. Data are plotted as mean±S.E.M. (n≥3). ( c ) TNF- α was measured in supernatants of 5FU-treated HCT.shC10 cells. Data are plotted as mean±S.E.M. ( n =3). ( d ) HCT.shC10 cells after 5FU treatment were analysed by western blot for I κ B- α and phospho-I κ B- α . ( e ) HCT.shC10 cells expressing I κ B-SR (Ad.I κ B-SR) were treated with 5FU, after which TNF- α and apoptosis were measured. Ad.EGFP served as control. Data are plotted as mean±S.E.M. ( n ≥3). ( f ) HCT116 cells silenced for both caspase-10 and caspase-8 were treated with 5FU and TNF- α was measured. Data are plotted as mean±S.E.M. ( n ≥5). ( g ) Western blot for cFLIP in HCT116 and HCT.shC10 cells after 5FU treatment. ( h ) EGFP, cFLIP S , cFLIP L , cFLIP p43 and cFLIP D376N (FLIP DN ) were expressed in HCT116 cells, and then the cells treated with 5FU before TNF- α levels were measured. Data are plotted as mean±S.E.M. ( n ≥2). ( i ) HCT.shC10 cells were silenced for cFLIP (Ad.shFLIP). TNF- α in cell culture supernatants was then determined after 5FU stimulation. Data are plotted as mean±S.E.M. ( n =2). ( j ) Either cFLIP L or cFLIP p43 were expressed in HCT.shC8 cells. After 5FU treatment, TNF- α was measured. Data are plotted as mean±S.E.M. (n≥3). ( k ) Precipitates from a caspase-8 IP were probed for cFLIP, FADD and caspase-8. The lysates were from HCT116 cells overexpressing cFLIP L and treated with 5FU. Cells overexpressing EGFP were used as controls. Input controls are shown on the left. ( l ) End point tumour volumes of HCT.shC10 xenografts treated with 5FU are depicted in relation to starting volumes (set to 100). (animal numbers/group: n =6/shctrl, n =3/shC10].

    Article Snippet: Sheep anti-CuZnSOD (The Binding Site, Birmingham, UK), goat and mouse anti-actin (Santa Cruz Biotechnology), rabbit anti-TRAIL-R2 (Cell Signaling Technology, Danvers, MA, USA), mouse anti-caspase-3 (Imgenex, Minneapolis, MN, USA), mouse anti-caspase-8 (Cell Signaling Technology, Santa Cruz Biotechnology and BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-XIAP (BD Biosciences), mouse anti-cFLIP (AdipoGen, San Diego, CA, USA), goat anti-cIAP1 (R & D Systems), rat anti-cIAP2 (Enzo Life Sciences), mouse anti-caspase-9 (Novus Biologicals, Minneapolis, MN, USA), mouse anti-caspase-2 (BD Biosciences), mouse anti-CD178 (BD Biosciences), mouse anti-TNFR1 (Santa Cruz Biotechnology), rabbit anti-TRAIL (Peprotech), rabbit anti-TRAIL-R1 (Santa Cruz Biotechnology), mouse anti-TNFR1 (Hycult Biotech, Uden, Netherlands), mouse anti-FADD (Millipore, Billerica, MA, USA), rabbit anti-Bid (R & D Systems), mouse anti-RIP1 (BD Biosciences), mouse anti-p53 (BD Biosciences), mouse anti-caspase-10 (MBL, Woburn, MA, USA), rabbit anti-CD95 (Santa Cruz Biotechnology), mouse anti-TRAF2 (Santa Cruz Biotechnology), rabbit anti-TRADD (Cell Signaling Technology), rabbit anti-CRADD/RAIDD (Cell Signaling Technology), rabbit anti-NEMO (Santa Cruz Biotechnology), mouse anti-Myc (Santa Cruz Biotechnology), mouse anti-V5 (Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-I κ B- α (Cell Signaling Technology), rabbit anti-Phospho-I κ B- α (Cell Signaling Technology), rabbit anti-Phospho-ATR (GeneTex, Irvine, CA, USA), rabbit anti-ATR (Bethyl Laboratories, Montgomery, TX, USA), rabbit anti-Phospho-ATM (Cell Signaling Technology), mouse anti-ATM (Sigma), rabbit anti-Phospho-Chk1 (Cell Signaling Technology), mouse anti-Chk1 (Cell Signaling Technology), mouse anti-TNF- α (Biolegend, San Diego, CA, USA), IgG1 Isotype antibody (BD Biosciences), IgG2a Isotype antibody (Biolegend), IgG2b Isotype antibody (Biolegend), mouse anti-Smac/DIABLO antibody (Abcam, Cambridge, UK), FITC-conjugated F(ab‘)2 anti-mouse IgG2a antibody (Southern Biotechnology, Birmingham, AL, USA), FITC-conjugated F(ab‘)2 anti-mouse IgG2b antibody (Southern Biotechnology), mouse anti-cytochrome c (BD Biosciences).

    Techniques: Activity Assay, Clone Assay, Blocking Assay, Western Blot, Expressing, Cell Culture

    Blocking effect of IL12 mAb on the suppressive effect of CpG on α-GalCer-induced AHR, airway inflammation, and cytokine production. As described in Fig. 3 , AHR to methacholine (A), inflammatory cells (B), and cytokines (C, D, and E) in BAL fluid were measured. IL-12 mAb or the IgG2b isotype was intraperitoneally administered immediately before CpG administration. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). * P

    Journal: Allergy, Asthma & Immunology Research

    Article Title: TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner

    doi: 10.4168/aair.2012.4.5.295

    Figure Lengend Snippet: Blocking effect of IL12 mAb on the suppressive effect of CpG on α-GalCer-induced AHR, airway inflammation, and cytokine production. As described in Fig. 3 , AHR to methacholine (A), inflammatory cells (B), and cytokines (C, D, and E) in BAL fluid were measured. IL-12 mAb or the IgG2b isotype was intraperitoneally administered immediately before CpG administration. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). * P

    Article Snippet: Cells were incubated with allophycocyanin-conjugated IL-4 (BD Biosciences) or IFN-γ mAbs (BD Biosciences), and the respective isotype IgG1 antibodies (BD Biosciences).

    Techniques: Blocking Assay

    Blocking effect of IL12 mAb on the suppressive effect of lipopolysaccharide (LPS) on α-GalCer-induced AHR, airway inflammation and cytokine production. As described in Fig. 1 , AHR to methacholine (A), inflammatory cells (B) and cytokines (C, D, and E) in BAL fluid were measured. IL-12 mAb or the IgG2b isotype was intraperitoneally administered immediately before LPS administration. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). * P

    Journal: Allergy, Asthma & Immunology Research

    Article Title: TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner

    doi: 10.4168/aair.2012.4.5.295

    Figure Lengend Snippet: Blocking effect of IL12 mAb on the suppressive effect of lipopolysaccharide (LPS) on α-GalCer-induced AHR, airway inflammation and cytokine production. As described in Fig. 1 , AHR to methacholine (A), inflammatory cells (B) and cytokines (C, D, and E) in BAL fluid were measured. IL-12 mAb or the IgG2b isotype was intraperitoneally administered immediately before LPS administration. Penh was expressed as the percentage increase in Penh, where Penh after PBS represented 100%. Total cells, Mac, Eos, Neut, and Lymph were expressed as the number per mL BAL fluid. Data are expressed as the mean±SEM (n=5). * P

    Article Snippet: Cells were incubated with allophycocyanin-conjugated IL-4 (BD Biosciences) or IFN-γ mAbs (BD Biosciences), and the respective isotype IgG1 antibodies (BD Biosciences).

    Techniques: Blocking Assay

    Effects of LPS, FlaB, or CpG on IL-4 and IFN-γ production from α-GalCer-stimulated spleen invariant natural killer T (iNKT) cells and the blocking effect of IL12 mAb. Spleen cells were stimulated with α-GalCer and, 90 min later, intracellular cytokines of iNKT cells were stained. LPS, FlaB, or CpG was added 16 hr before α-GalCer stimulation. IL-12 or the IgG2b isotype was added immediately before addition of LPS, FlaB, or CpG. CD3+ α-GalCer-loaded CD1d tetramer+ iNKT cells were gated and the proportion of IL-4+ or IFN-γ + iNKT cells was analyzed, in which IgG1 isotypes were used for IL-4 and IFN-γ mAbs (A). IL-4+ (B) and IFN-γ + (C) iNKT cells were measured in cultures treated with LPS. IL-4+ (D) and IFN-γ + (E) iNKT cells were measured in cultures treated with FlaB. IL-4+ (F) and IFN-γ + (G) iNKT cells were measured in cultures treated with CpG. Spleen cells were pooled from three mice, and data are expressed as the mean±SEM of triplicate measurements. * P

    Journal: Allergy, Asthma & Immunology Research

    Article Title: TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner

    doi: 10.4168/aair.2012.4.5.295

    Figure Lengend Snippet: Effects of LPS, FlaB, or CpG on IL-4 and IFN-γ production from α-GalCer-stimulated spleen invariant natural killer T (iNKT) cells and the blocking effect of IL12 mAb. Spleen cells were stimulated with α-GalCer and, 90 min later, intracellular cytokines of iNKT cells were stained. LPS, FlaB, or CpG was added 16 hr before α-GalCer stimulation. IL-12 or the IgG2b isotype was added immediately before addition of LPS, FlaB, or CpG. CD3+ α-GalCer-loaded CD1d tetramer+ iNKT cells were gated and the proportion of IL-4+ or IFN-γ + iNKT cells was analyzed, in which IgG1 isotypes were used for IL-4 and IFN-γ mAbs (A). IL-4+ (B) and IFN-γ + (C) iNKT cells were measured in cultures treated with LPS. IL-4+ (D) and IFN-γ + (E) iNKT cells were measured in cultures treated with FlaB. IL-4+ (F) and IFN-γ + (G) iNKT cells were measured in cultures treated with CpG. Spleen cells were pooled from three mice, and data are expressed as the mean±SEM of triplicate measurements. * P

    Article Snippet: Cells were incubated with allophycocyanin-conjugated IL-4 (BD Biosciences) or IFN-γ mAbs (BD Biosciences), and the respective isotype IgG1 antibodies (BD Biosciences).

    Techniques: Blocking Assay, Staining, Mouse Assay

    Histone kills Leishmania . ( A ) Killing of Leishmania by NETs is inhibited by antihistone antibody. Neutrophils treated with PMA and CytD were exposed to 5 μg/mL of anti-H2A histone or IgG isotypic antibodies, and then incubated with promastigotes (1 cell/0.1 parasite ratio) for 2 h at 35 °C. Schneider's complete medium was added to the cultures and live parasites were counted after 2 days incubation at 26 °C. Results of 4 independent experiments are shown as mean + SEM. PMA raw number: 6.6 × 10 5 + 0.8 × 10 5 promastigotes. *, P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps

    doi: 10.1073/pnas.0900226106

    Figure Lengend Snippet: Histone kills Leishmania . ( A ) Killing of Leishmania by NETs is inhibited by antihistone antibody. Neutrophils treated with PMA and CytD were exposed to 5 μg/mL of anti-H2A histone or IgG isotypic antibodies, and then incubated with promastigotes (1 cell/0.1 parasite ratio) for 2 h at 35 °C. Schneider's complete medium was added to the cultures and live parasites were counted after 2 days incubation at 26 °C. Results of 4 independent experiments are shown as mean + SEM. PMA raw number: 6.6 × 10 5 + 0.8 × 10 5 promastigotes. *, P

    Article Snippet: In some experiments, neutrophil and promastigote cocultures were incubated either with 10 μg/mL of the neutrophil elastase inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (Calbiochem), 5 μg/mL of the antihistone H2A monoclonal antibody (donated by A. Zychlinsky, Max Planck Institute, Berlin), or IgG isotype control (Sigma) in the interaction medium.

    Techniques: Incubation

    Antibody isotype patterns. Patterns of induction of anti-S IgG isotypes were compared after three immunizations with alum-based or L-pampo-based L-HBsAg formulations. Each formulation contained 0.5 μg of L-HBsAg. L-HBsAg formulated with L-pampo

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Use of Pre-S Protein-Containing Hepatitis B Virus Surface Antigens and a Powerful Adjuvant To Develop an Immune Therapy for Chronic Hepatitis B Virus Infection

    doi: 10.1128/CVI.05355-11

    Figure Lengend Snippet: Antibody isotype patterns. Patterns of induction of anti-S IgG isotypes were compared after three immunizations with alum-based or L-pampo-based L-HBsAg formulations. Each formulation contained 0.5 μg of L-HBsAg. L-HBsAg formulated with L-pampo

    Article Snippet: To determine the titers of antibody isotypes IgG1, IgG2a, and IgG2b, mouse monoclonal antibody isotyping reagents (Sigma) were used.

    Techniques: