anti-mouse igg Search Results


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    99
    Vector Laboratories biotinylated secondary antibodies
    Biotinylated Secondary Antibodies, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 10949 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg h l cross adsorbed secondary antibody
    Goat Anti Mouse Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg h l highly cross adsorbed secondary antibody
    Goat Anti Mouse Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9071 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher donkey anti mouse igg h l highly cross adsorbed secondary antibody
    Donkey Anti Mouse Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6700 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti mouse igg
    Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti mouse igg whole molecule peroxidase antibody
    Anti Mouse Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg
    Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9641 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology goat anti mouse igg hrp
    Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat <t>IgG-HRP</t> (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P
    Goat Anti Mouse Igg Hrp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 4245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti mouse igg
    Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat <t>IgG-HRP</t> (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P
    Anti Mouse Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4035 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno peroxidase affinipure goat anti mouse igg
    Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat <t>IgG-HRP</t> (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P
    Peroxidase Affinipure Goat Anti Mouse Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 3302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti rabbit igg
    Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat <t>IgG-HRP</t> (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P
    Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LI-COR irdye 800cw goat anti mouse igg h l
    Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat <t>IgG-HRP</t> (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P
    Irdye 800cw Goat Anti Mouse Igg H L, supplied by LI-COR, used in various techniques. Bioz Stars score: 99/100, based on 2660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti mouse igg
    Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat <t>IgG-HRP</t> (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P
    Anti Mouse Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4693 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories biotinylated anti mouse igg
    Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat <t>IgG-HRP</t> (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P
    Biotinylated Anti Mouse Igg, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 3254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology goat anti mouse igg
    <t>HCt/E-specific</t> antibody production in mice immunized with BoNT/HCt/E DNA vaccine by intradermal electroporation. Mice were immunized 3 times (week 0, 2, 4) with 50 μg of DNA vaccine. Anti-HCt/E <t>IgG</t> levels are expressed as endpoint titers. Each symbol represents data from an individual mouse. The bar represents the mean titer for each group.
    Goat Anti Mouse Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2624 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti mouse secondary antibody
    <t>HCt/E-specific</t> antibody production in mice immunized with BoNT/HCt/E DNA vaccine by intradermal electroporation. Mice were immunized 3 times (week 0, 2, 4) with 50 μg of DNA vaccine. Anti-HCt/E <t>IgG</t> levels are expressed as endpoint titers. Each symbol represents data from an individual mouse. The bar represents the mean titer for each group.
    Anti Mouse Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2084 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti mouse igg
    PKR both interacts with MAVS and <t>TRAF3</t> and binds HCV RNA ahead of RIG-I. ( A–B )- Huh7.25.CD81 cells were transfected with 25 nM of siRNA Control ( A ) or 25 nM of siRNA ISG15 ( B ) for 48 hrs and infected with JFH1 (m.o.i = 0.2). At the times indicated, cell extracts (4.5 mg) were incubated with anti-MAVS antibodies. In addition, cell extracts prepared at 0 hr post-infection were incubated with mouse <t>IgG</t> as a control of specificity (asterisk). The immunoprecipitated complexes were run on three different NuPAGE gels and blotted using Mab 71/10, anti-MAVS, anti-RIG-I or anti-TRAF3 antibodies. The expression level of each protein was controlled in the total cell extracts. ( C )- Huh7.25.CD81 cells were incubated with JFH1 (m.o.i = 6) for 2 hrs at 37°C or at 4°C in the absence or presence of 30 µM of PRI. This drug was applied one hour before the end of the incubation time. After washing the cells twice with PBS, the cells were further incubated for 2, 4 or 6 hrs at 37°C or at 4°C in the absence or presence of PRI (added every hour). The cell extracts were processed for crosslinking of RNA to proteins before lysis, as described in Materials and Methods and different immunoprecipitations were performed with antibodies directed against PKR, RIG-I or eIF2α. After extensive washing, the presence of HCV RNA linked to the immunocomplexes was analysed by RTqPCR and the presence of the proteins was verified by Western blot. Measure of HCV RNA in the cell extracts allowed to estimate its percentage of binding to PKR as 1.09%, 0.47% and 0.25% at 2, 4 and 6 hrs post-infection respectively, and its percentage of binding to RIG-I as 0.34% at 6 hrs post-infection.
    Anti Mouse Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 3062 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SouthernBiotech goat anti mouse igg1
    Induction of anti-HA humoral and cellular responses in mice vaccinated with VLP-HA/VSP-G. a – e Mice were orally vaccinated with VLPs and the humoral response against HA in different biological samples was evaluated. Levels of total serum anti-HA IgG ( a ), <t>IgG1</t> and IgG2a ( b ), n = 8 from two independent experiments. Levels of IgA anti-HA in fecal extracts and BAL, n = 6 from two independent experiments ( c ). Levels of serum IgG anti-HA measured at different times post immunization, n = 6 from two independent experiments ( d ). Levels of serum IgG anti-HA in animals immunized with a mixture of VLP-HA plus VLP-VSP-G, n = 6 from two independent experiments ( e ). f Levels of serum IgG and fecal IgA in mice vaccinated with VLPs via oral or subcutaneous (s.c.) route, n = 8 from two independent experiments. g Mice were orally immunized with four weekly doses of 50 μg of recombinant ΔVSP1267 or vehicle. One week after the last immunization the presence of anti-VSP1267 antibodies in blood and fecal samples was checked, then these animals were orally immunized with VLP-HA/VSP-G, according to the protocol previously used. One week after the last VLP dose, the levels of serum IgG and fecal IgA anti-HA Abs were measured, n = 6 from two independent experiments. h , i Mice were orally vaccinated with VLPs and the cellular response against HA was evaluated. The frequency of HA-specific IFN-γ-secreting T cells (spot-forming colonies, SFC) was determined after antigen-specific re-stimulation of splenocytes, n = 7 from two independent experiments ( h ). Cytokines were measured in splenocyte supernatants and only those cytokines with detectable levels are shown, n = 6 from two independent experiments ( i ). Data were analyzed by one-way ANOVA and Tukey’s multiple comparison test ( a – f , h , i ) or by two-way ANOVA and Bonferroni post-tests ( g ). Values represent mean ± s.e.m. * p
    Goat Anti Mouse Igg1, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 99/100, based on 707 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher igg
    Induction of anti-HA humoral and cellular responses in mice vaccinated with VLP-HA/VSP-G. a – e Mice were orally vaccinated with VLPs and the humoral response against HA in different biological samples was evaluated. Levels of total serum anti-HA IgG ( a ), <t>IgG1</t> and IgG2a ( b ), n = 8 from two independent experiments. Levels of IgA anti-HA in fecal extracts and BAL, n = 6 from two independent experiments ( c ). Levels of serum IgG anti-HA measured at different times post immunization, n = 6 from two independent experiments ( d ). Levels of serum IgG anti-HA in animals immunized with a mixture of VLP-HA plus VLP-VSP-G, n = 6 from two independent experiments ( e ). f Levels of serum IgG and fecal IgA in mice vaccinated with VLPs via oral or subcutaneous (s.c.) route, n = 8 from two independent experiments. g Mice were orally immunized with four weekly doses of 50 μg of recombinant ΔVSP1267 or vehicle. One week after the last immunization the presence of anti-VSP1267 antibodies in blood and fecal samples was checked, then these animals were orally immunized with VLP-HA/VSP-G, according to the protocol previously used. One week after the last VLP dose, the levels of serum IgG and fecal IgA anti-HA Abs were measured, n = 6 from two independent experiments. h , i Mice were orally vaccinated with VLPs and the cellular response against HA was evaluated. The frequency of HA-specific IFN-γ-secreting T cells (spot-forming colonies, SFC) was determined after antigen-specific re-stimulation of splenocytes, n = 7 from two independent experiments ( h ). Cytokines were measured in splenocyte supernatants and only those cytokines with detectable levels are shown, n = 6 from two independent experiments ( i ). Data were analyzed by one-way ANOVA and Tukey’s multiple comparison test ( a – f , h , i ) or by two-way ANOVA and Bonferroni post-tests ( g ). Values represent mean ± s.e.m. * p
    Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9957 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg1 cross adsorbed secondary antibody
    Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP <t>IgG1</t> in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test
    Goat Anti Mouse Igg1 Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories biotinylated horse anti mouse igg
    Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP <t>IgG1</t> in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test
    Biotinylated Horse Anti Mouse Igg, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 2568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories biotinylated antibody
    Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP <t>IgG1</t> in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test
    Biotinylated Antibody, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 1769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Rabbit polyclonal to IgG1 IgG2a IgG2b IgG3 TRITC Isotype Note IgG Host Note Rabbit Conjugation Note TRITC Reactivity Note Mouse Application Note ELISA IHC P IHC Fr IF ICC
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    Horseradish peroxidase conjugated IgG fraction of polyclonal rabbit antiserum to Mouse IgG Fc specific In enzyme immunocytochemical and immunohistochemical staining for the detection of IgG at the cellular and subcellular
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    Monoclonal Mouse antibody to Human IgG3 subclass specific Isotype Note IgG1 Host Species Note Mouse Reactivity Note The antiSerum does not react with any other component of the Human Ig
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    Image Search Results


    Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat IgG-HRP (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P

    Journal: Oncology Reports

    Article Title: Increased trefoil factor 3 levels in the serum of patients with three major histological subtypes of lung cancer

    doi: 10.3892/or.2012.1627

    Figure Lengend Snippet: Immunoblots of TFF1, TFF2 and TFF3 in a normal cell line and three lung cancer cell lines. (A) Total proteins were harvested, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat IgG-HRP (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins were approximately 7–10 kDa. Experiments were repeated more than 3 times. Histograms show mean normalized OD of TFF protein bands relative to the OD of actin band. Error bars show standard error of the mean (SEM) (P

    Article Snippet: Secondary antibodies used in this study were donkey anti-goat IgG-HRP (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (Cat# sc-2005, 1:10,000, Santa Cruz Biotechnology).

    Techniques: Western Blot, SDS Page

    Immunoblots of TFF1, TFF2 and TFF3 in healthy individuals and lung cancer patients. (A) Total proteins were extracted from lung tissues, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat IgG-HRP (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins was approximately 7–10 kDa. Histograms show mean normalized optical density (OD) of TFF protein bands relative to the OD of the actin band from the same individual. Error bars show the standard error of the mean (SEM) (P

    Journal: Oncology Reports

    Article Title: Increased trefoil factor 3 levels in the serum of patients with three major histological subtypes of lung cancer

    doi: 10.3892/or.2012.1627

    Figure Lengend Snippet: Immunoblots of TFF1, TFF2 and TFF3 in healthy individuals and lung cancer patients. (A) Total proteins were extracted from lung tissues, separated on SDS-PAGE gels, and subjected to immunoblot analyses. The primary antibodies against TFF1, TFF2, TFF3 and actin were purchased from Santa Cruz Biotechnology (USA). Secondary antibodies were donkey anti-goat IgG-HRP (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The size of the TFF proteins was approximately 7–10 kDa. Histograms show mean normalized optical density (OD) of TFF protein bands relative to the OD of the actin band from the same individual. Error bars show the standard error of the mean (SEM) (P

    Article Snippet: Secondary antibodies used in this study were donkey anti-goat IgG-HRP (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (Cat# sc-2005, 1:10,000, Santa Cruz Biotechnology).

    Techniques: Western Blot, SDS Page

    HCt/E-specific antibody production in mice immunized with BoNT/HCt/E DNA vaccine by intradermal electroporation. Mice were immunized 3 times (week 0, 2, 4) with 50 μg of DNA vaccine. Anti-HCt/E IgG levels are expressed as endpoint titers. Each symbol represents data from an individual mouse. The bar represents the mean titer for each group.

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: Intradermal immunization with botulinum neurotoxin serotype E DNA vaccine induces humoral and cellular immunity and protects against lethal toxin challenge

    doi: 10.1080/21645515.2018.1526554

    Figure Lengend Snippet: HCt/E-specific antibody production in mice immunized with BoNT/HCt/E DNA vaccine by intradermal electroporation. Mice were immunized 3 times (week 0, 2, 4) with 50 μg of DNA vaccine. Anti-HCt/E IgG levels are expressed as endpoint titers. Each symbol represents data from an individual mouse. The bar represents the mean titer for each group.

    Article Snippet: The HCt/E protein was detected using a mouse anti-HCt/E antibody and goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotech, Santa Cruz, USA).

    Techniques: Mouse Assay, Electroporation

    Survival and antibody titers following immunization with HCt/E protein vaccine. Mice were immunized three times at 2-week intervals with 10 μg or 20 μg of purified HCt/E vaccine intramuscularly, followed by intraperitoneal challenge with 200 LD 50 of active BoNT/E. The number of survivors are shown. Anti-HCt/E IgG levels are expressed as endpoint titers. Sera were obtained one week after last immunization. Each symbol represents data from an individual mouse. The bar represents the mean titer for each group.

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: Intradermal immunization with botulinum neurotoxin serotype E DNA vaccine induces humoral and cellular immunity and protects against lethal toxin challenge

    doi: 10.1080/21645515.2018.1526554

    Figure Lengend Snippet: Survival and antibody titers following immunization with HCt/E protein vaccine. Mice were immunized three times at 2-week intervals with 10 μg or 20 μg of purified HCt/E vaccine intramuscularly, followed by intraperitoneal challenge with 200 LD 50 of active BoNT/E. The number of survivors are shown. Anti-HCt/E IgG levels are expressed as endpoint titers. Sera were obtained one week after last immunization. Each symbol represents data from an individual mouse. The bar represents the mean titer for each group.

    Article Snippet: The HCt/E protein was detected using a mouse anti-HCt/E antibody and goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotech, Santa Cruz, USA).

    Techniques: Mouse Assay, Purification

    Survival and antibody titers following immunization with BoNT/HCt/E DNA vaccine. Mice were immunized two or three times at 2-week intervals with various doses of DNA vaccine intradermally via electroporation, followed by intraperitoneal challenge with 50 LD 50 of active BoNT/E. (A) Survival curves show the percentage of mice alive after challenge. Data shown are from one of two experiments. (B) Anti-HCt/E IgG levels are expressed as endpoint titers. Sera were obtained one week before challenged. Each symbol represents data from an individual mouse. The bar represents the mean titer for each group. Control group mice were immunized with vector only. Significant results are marked asterisks: ** p

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: Intradermal immunization with botulinum neurotoxin serotype E DNA vaccine induces humoral and cellular immunity and protects against lethal toxin challenge

    doi: 10.1080/21645515.2018.1526554

    Figure Lengend Snippet: Survival and antibody titers following immunization with BoNT/HCt/E DNA vaccine. Mice were immunized two or three times at 2-week intervals with various doses of DNA vaccine intradermally via electroporation, followed by intraperitoneal challenge with 50 LD 50 of active BoNT/E. (A) Survival curves show the percentage of mice alive after challenge. Data shown are from one of two experiments. (B) Anti-HCt/E IgG levels are expressed as endpoint titers. Sera were obtained one week before challenged. Each symbol represents data from an individual mouse. The bar represents the mean titer for each group. Control group mice were immunized with vector only. Significant results are marked asterisks: ** p

    Article Snippet: The HCt/E protein was detected using a mouse anti-HCt/E antibody and goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotech, Santa Cruz, USA).

    Techniques: Mouse Assay, Electroporation, Plasmid Preparation

    PKR both interacts with MAVS and TRAF3 and binds HCV RNA ahead of RIG-I. ( A–B )- Huh7.25.CD81 cells were transfected with 25 nM of siRNA Control ( A ) or 25 nM of siRNA ISG15 ( B ) for 48 hrs and infected with JFH1 (m.o.i = 0.2). At the times indicated, cell extracts (4.5 mg) were incubated with anti-MAVS antibodies. In addition, cell extracts prepared at 0 hr post-infection were incubated with mouse IgG as a control of specificity (asterisk). The immunoprecipitated complexes were run on three different NuPAGE gels and blotted using Mab 71/10, anti-MAVS, anti-RIG-I or anti-TRAF3 antibodies. The expression level of each protein was controlled in the total cell extracts. ( C )- Huh7.25.CD81 cells were incubated with JFH1 (m.o.i = 6) for 2 hrs at 37°C or at 4°C in the absence or presence of 30 µM of PRI. This drug was applied one hour before the end of the incubation time. After washing the cells twice with PBS, the cells were further incubated for 2, 4 or 6 hrs at 37°C or at 4°C in the absence or presence of PRI (added every hour). The cell extracts were processed for crosslinking of RNA to proteins before lysis, as described in Materials and Methods and different immunoprecipitations were performed with antibodies directed against PKR, RIG-I or eIF2α. After extensive washing, the presence of HCV RNA linked to the immunocomplexes was analysed by RTqPCR and the presence of the proteins was verified by Western blot. Measure of HCV RNA in the cell extracts allowed to estimate its percentage of binding to PKR as 1.09%, 0.47% and 0.25% at 2, 4 and 6 hrs post-infection respectively, and its percentage of binding to RIG-I as 0.34% at 6 hrs post-infection.

    Journal: PLoS Pathogens

    Article Title: Hepatitis C Virus Reveals a Novel Early Control in Acute Immune Response

    doi: 10.1371/journal.ppat.1002289

    Figure Lengend Snippet: PKR both interacts with MAVS and TRAF3 and binds HCV RNA ahead of RIG-I. ( A–B )- Huh7.25.CD81 cells were transfected with 25 nM of siRNA Control ( A ) or 25 nM of siRNA ISG15 ( B ) for 48 hrs and infected with JFH1 (m.o.i = 0.2). At the times indicated, cell extracts (4.5 mg) were incubated with anti-MAVS antibodies. In addition, cell extracts prepared at 0 hr post-infection were incubated with mouse IgG as a control of specificity (asterisk). The immunoprecipitated complexes were run on three different NuPAGE gels and blotted using Mab 71/10, anti-MAVS, anti-RIG-I or anti-TRAF3 antibodies. The expression level of each protein was controlled in the total cell extracts. ( C )- Huh7.25.CD81 cells were incubated with JFH1 (m.o.i = 6) for 2 hrs at 37°C or at 4°C in the absence or presence of 30 µM of PRI. This drug was applied one hour before the end of the incubation time. After washing the cells twice with PBS, the cells were further incubated for 2, 4 or 6 hrs at 37°C or at 4°C in the absence or presence of PRI (added every hour). The cell extracts were processed for crosslinking of RNA to proteins before lysis, as described in Materials and Methods and different immunoprecipitations were performed with antibodies directed against PKR, RIG-I or eIF2α. After extensive washing, the presence of HCV RNA linked to the immunocomplexes was analysed by RTqPCR and the presence of the proteins was verified by Western blot. Measure of HCV RNA in the cell extracts allowed to estimate its percentage of binding to PKR as 1.09%, 0.47% and 0.25% at 2, 4 and 6 hrs post-infection respectively, and its percentage of binding to RIG-I as 0.34% at 6 hrs post-infection.

    Article Snippet: Other antibodies were as follows: anti-mouse IgG (Santa Cruz), anti-TRAF3 (Santa Cruz), pThr451-PKR (Alexis), MAVS (Alexis), anti-actin (Sigma), anti-pSer10-Histone H3 (Millipore), anti-HCV NS3 (Chemicon), anti-HCV core (Thermo scientific), anti-RIG-I (Alexis Biochemical Inc.), anti-TRIM25 (6105710; BD Bioscience), anti-IRF3 (Santa Cruz), anti-HA (12CA5; Roche) and anti-Myc (Santa Cruz).

    Techniques: Transfection, Infection, Incubation, Immunoprecipitation, Expressing, Lysis, Western Blot, Binding Assay

    ISG15 strengthens the pro-HCV activity of PKR. ( A–B ). The Huh7.25.CD81 cells were transfected with 25 nM of the different siRNA (Control, ISG15, PKR), separately or together. After 48 hrs, cells were infected with JFH1 (m.o.i = 0.2). At the times indicated, expression of HCV or IFNβ RNA was determined by RTqPCR and expressed as copies of JFH1 RNA ( A ) or as fold induction (IFNβ; B ). The expression levels of IFNβ RNA at the start of infection was 6.96×10 5 copies. ( C ) Two sets of Huh7.25.CD81 cells were first transfected with siRNA ISG15, siRNA PKR separately and together for 24 hrs, then transfected with the reporter plasmids IFNβ-firefly luciferase (pGL2-IFNβ), pRL-TK Renilla-luciferase for another 24 hrs and infected with JFH1 (m.o.i = 0.2) for the times indicated. In each case, IFN expression was expressed as fold-induction over control cells that were simply transfected with pGL2-IFNβ-FLUC/pRL-TK-RLUC. The graph represents the level of firefly luciferase activity normalized to the ratio R-luc RNA/GAPDH RNA. Such normalization is required because of the negative control of general translation through PKR after 12 hrs post-infection [8] . Error bars represent the mean ±S.D for triplicates. ( D ) Huh7.25.CD81 cells, in 100 cm 2 plates, were transfected with siRNA Control or siRNA ISG15 or transfected with a plasmid expressing HA-ISG15 for 48 hrs and infected with JFH1 (m.o.i = 6). At the indicated times post-infection, cell extracts (2.2 mg) were processed for immunoprecipitation of PKR or for incubation with mouse IgG as a control of specificity (asterisk). The immunoprecipitated complexes were run on two different NuPAGE gels and blotted using Mab 71/10 or anti-phosphorylated PKR antibodies (PKR-P). The presence of PKR and PKR-P was revealed using the Odyssey procedure. The ratio PKR-P/PKR in the absence or in the presence of ISG15, either endogenous or endogenous and ectopic, is shown in Figure S5 .

    Journal: PLoS Pathogens

    Article Title: Hepatitis C Virus Reveals a Novel Early Control in Acute Immune Response

    doi: 10.1371/journal.ppat.1002289

    Figure Lengend Snippet: ISG15 strengthens the pro-HCV activity of PKR. ( A–B ). The Huh7.25.CD81 cells were transfected with 25 nM of the different siRNA (Control, ISG15, PKR), separately or together. After 48 hrs, cells were infected with JFH1 (m.o.i = 0.2). At the times indicated, expression of HCV or IFNβ RNA was determined by RTqPCR and expressed as copies of JFH1 RNA ( A ) or as fold induction (IFNβ; B ). The expression levels of IFNβ RNA at the start of infection was 6.96×10 5 copies. ( C ) Two sets of Huh7.25.CD81 cells were first transfected with siRNA ISG15, siRNA PKR separately and together for 24 hrs, then transfected with the reporter plasmids IFNβ-firefly luciferase (pGL2-IFNβ), pRL-TK Renilla-luciferase for another 24 hrs and infected with JFH1 (m.o.i = 0.2) for the times indicated. In each case, IFN expression was expressed as fold-induction over control cells that were simply transfected with pGL2-IFNβ-FLUC/pRL-TK-RLUC. The graph represents the level of firefly luciferase activity normalized to the ratio R-luc RNA/GAPDH RNA. Such normalization is required because of the negative control of general translation through PKR after 12 hrs post-infection [8] . Error bars represent the mean ±S.D for triplicates. ( D ) Huh7.25.CD81 cells, in 100 cm 2 plates, were transfected with siRNA Control or siRNA ISG15 or transfected with a plasmid expressing HA-ISG15 for 48 hrs and infected with JFH1 (m.o.i = 6). At the indicated times post-infection, cell extracts (2.2 mg) were processed for immunoprecipitation of PKR or for incubation with mouse IgG as a control of specificity (asterisk). The immunoprecipitated complexes were run on two different NuPAGE gels and blotted using Mab 71/10 or anti-phosphorylated PKR antibodies (PKR-P). The presence of PKR and PKR-P was revealed using the Odyssey procedure. The ratio PKR-P/PKR in the absence or in the presence of ISG15, either endogenous or endogenous and ectopic, is shown in Figure S5 .

    Article Snippet: Other antibodies were as follows: anti-mouse IgG (Santa Cruz), anti-TRAF3 (Santa Cruz), pThr451-PKR (Alexis), MAVS (Alexis), anti-actin (Sigma), anti-pSer10-Histone H3 (Millipore), anti-HCV NS3 (Chemicon), anti-HCV core (Thermo scientific), anti-RIG-I (Alexis Biochemical Inc.), anti-TRIM25 (6105710; BD Bioscience), anti-IRF3 (Santa Cruz), anti-HA (12CA5; Roche) and anti-Myc (Santa Cruz).

    Techniques: Activity Assay, Transfection, Infection, Expressing, Luciferase, Negative Control, Plasmid Preparation, Immunoprecipitation, Incubation

    HCV-dependent induction of ISG15 involves PKR, MAVS and TRAF3 and not RIG-I. ( A–B ). A -The Huh7.25.CD81 cells were transfected with 50 nM Control siRNA and the different Smartpool siRNAs (50 nM siPKR; 10 nM siRIG-I; 5 nM si MAVS; 50 nM siTRAF3; 50 nM siIRF3) for 48 hrs and infected with JFH1 (m.o.i = 6). ( B ) Huh7.5 or Huh7 cells were transfected with siRNA Control or siPKR (50 nM) for 48 hrs and infected with JFH1 (m.o.i = 0.2 for Huh7.5 or 10 for Huh7). At the times indicated, expression of endogenous ISG15 was determined by RTqPCR and expressed as fold induction. Error bars represent the mean ±S.D for triplicates. The expression level of ISG15 RNA at the start of infection in the siControl cells was 9.97×10 4 copies (Huh7.25.CD81), 1.31×10 4 copies (Huh7.5) and 1.28×10 4 (Huh7). ( C–D ) Huh7.25.CD81 cells, in 100 cm 2 plates, were infected with JFH1 (m.o.i = 0.2) alone ( C ) or in presence of PRI or C16 ( D ). At the times indicated, cell extracts (3.5 mg) were processed for immunoprecipitation of PKR or for incubation with mouse IgG as a control of specificity (asterisk). The detection of the proteins in the complexes and in the whole cell extracts (WCE) was revealed by immunoblot using the Odyssey procedure. ( E ) The Huh7.25.CD81 cells were incubated with PRI or C16 and infected with JFH1 (m.o.i = 0.2) for the times indicated. Expression of endogenous ISG15 was determined as in A–B. The ISG15 RNA levels were 3.81×10 4 copies in the siControl cells. ( F ) Human primary hepatocytes (HHP) were infected with JFH1 (m.o.i = 6). One set of cells was incubated with 30 mM of the PRI inhibitor during 8 hours. At the times indicated, expression of HCV RNA, ISG15 and IFNβ was determined by RTqPCR. The expression levels of ISG15 and IFNβ RNA at the start of infection was 1.05×10 5 copies and 1,11×10 4 copies, respectively. Inhibition of induction of ISG15 by PRI at 8 hr post-infection was statistically significant (***; p = 0.0001).

    Journal: PLoS Pathogens

    Article Title: Hepatitis C Virus Reveals a Novel Early Control in Acute Immune Response

    doi: 10.1371/journal.ppat.1002289

    Figure Lengend Snippet: HCV-dependent induction of ISG15 involves PKR, MAVS and TRAF3 and not RIG-I. ( A–B ). A -The Huh7.25.CD81 cells were transfected with 50 nM Control siRNA and the different Smartpool siRNAs (50 nM siPKR; 10 nM siRIG-I; 5 nM si MAVS; 50 nM siTRAF3; 50 nM siIRF3) for 48 hrs and infected with JFH1 (m.o.i = 6). ( B ) Huh7.5 or Huh7 cells were transfected with siRNA Control or siPKR (50 nM) for 48 hrs and infected with JFH1 (m.o.i = 0.2 for Huh7.5 or 10 for Huh7). At the times indicated, expression of endogenous ISG15 was determined by RTqPCR and expressed as fold induction. Error bars represent the mean ±S.D for triplicates. The expression level of ISG15 RNA at the start of infection in the siControl cells was 9.97×10 4 copies (Huh7.25.CD81), 1.31×10 4 copies (Huh7.5) and 1.28×10 4 (Huh7). ( C–D ) Huh7.25.CD81 cells, in 100 cm 2 plates, were infected with JFH1 (m.o.i = 0.2) alone ( C ) or in presence of PRI or C16 ( D ). At the times indicated, cell extracts (3.5 mg) were processed for immunoprecipitation of PKR or for incubation with mouse IgG as a control of specificity (asterisk). The detection of the proteins in the complexes and in the whole cell extracts (WCE) was revealed by immunoblot using the Odyssey procedure. ( E ) The Huh7.25.CD81 cells were incubated with PRI or C16 and infected with JFH1 (m.o.i = 0.2) for the times indicated. Expression of endogenous ISG15 was determined as in A–B. The ISG15 RNA levels were 3.81×10 4 copies in the siControl cells. ( F ) Human primary hepatocytes (HHP) were infected with JFH1 (m.o.i = 6). One set of cells was incubated with 30 mM of the PRI inhibitor during 8 hours. At the times indicated, expression of HCV RNA, ISG15 and IFNβ was determined by RTqPCR. The expression levels of ISG15 and IFNβ RNA at the start of infection was 1.05×10 5 copies and 1,11×10 4 copies, respectively. Inhibition of induction of ISG15 by PRI at 8 hr post-infection was statistically significant (***; p = 0.0001).

    Article Snippet: Other antibodies were as follows: anti-mouse IgG (Santa Cruz), anti-TRAF3 (Santa Cruz), pThr451-PKR (Alexis), MAVS (Alexis), anti-actin (Sigma), anti-pSer10-Histone H3 (Millipore), anti-HCV NS3 (Chemicon), anti-HCV core (Thermo scientific), anti-RIG-I (Alexis Biochemical Inc.), anti-TRIM25 (6105710; BD Bioscience), anti-IRF3 (Santa Cruz), anti-HA (12CA5; Roche) and anti-Myc (Santa Cruz).

    Techniques: Transfection, Infection, Expressing, Immunoprecipitation, Incubation, Inhibition

    Identification of nucleolin target mRNAs by RNP IP and microarray analysis. ( A ) IP assay using 3 mg of protein lysate prepared from HeLa cells and 20 μg of either anti-nucleolin antibody or IgG under conditions that preserved mRNP complexes. ( B ) Visualization of proteins present in the anti-nucleolin or IgG IP reactions, including nucleolin (arrowhead) and several unidentified proteins (asterisks). ( C ) Nucleolin was detected by western blot analysis in aliquots of IP samples. ( D ) Partial list of nucleolin target genes identified by microarray analysis. The threshold considered was ≥2-fold. For a complete list, see Supplementary Table S1 . ( E ) Nucleolin target mRNAs were analyzed using Ingenuity Pathway Analysis (IPA). The top target transcripts are involved in cancer, infection, cell growth and proliferation. In (B and D), ‘MW’, molecular weight markers (kDa).

    Journal: Nucleic Acids Research

    Article Title: Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs

    doi: 10.1093/nar/gkr488

    Figure Lengend Snippet: Identification of nucleolin target mRNAs by RNP IP and microarray analysis. ( A ) IP assay using 3 mg of protein lysate prepared from HeLa cells and 20 μg of either anti-nucleolin antibody or IgG under conditions that preserved mRNP complexes. ( B ) Visualization of proteins present in the anti-nucleolin or IgG IP reactions, including nucleolin (arrowhead) and several unidentified proteins (asterisks). ( C ) Nucleolin was detected by western blot analysis in aliquots of IP samples. ( D ) Partial list of nucleolin target genes identified by microarray analysis. The threshold considered was ≥2-fold. For a complete list, see Supplementary Table S1 . ( E ) Nucleolin target mRNAs were analyzed using Ingenuity Pathway Analysis (IPA). The top target transcripts are involved in cancer, infection, cell growth and proliferation. In (B and D), ‘MW’, molecular weight markers (kDa).

    Article Snippet: For microarray analysis, 3 mg of lysate were incubated (1 h, 4°C) with 100 ml of a 50% (v/v) suspension of protein-A Sepharose beads precoated with 20 mg each of mouse anti-nucleolin or mouse IgG (Santa Cruz).

    Techniques: Microarray, Western Blot, Indirect Immunoperoxidase Assay, Infection, Molecular Weight

    Validation of nucleolin targets by RT–qPCR. The abundance of transcripts present in material obtained from HeLa cells after either nucleolin IP or IgG IP was assessed by RT–qPCR analysis. Values show the mean enrichment + standard deviation (SD) from three independent experiments. Measurement of GAPDH mRNA served as loading and background control, since the highly abundant transcript (not a target of nucleolin) is a detectable contaminant present in the IP reactions.

    Journal: Nucleic Acids Research

    Article Title: Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs

    doi: 10.1093/nar/gkr488

    Figure Lengend Snippet: Validation of nucleolin targets by RT–qPCR. The abundance of transcripts present in material obtained from HeLa cells after either nucleolin IP or IgG IP was assessed by RT–qPCR analysis. Values show the mean enrichment + standard deviation (SD) from three independent experiments. Measurement of GAPDH mRNA served as loading and background control, since the highly abundant transcript (not a target of nucleolin) is a detectable contaminant present in the IP reactions.

    Article Snippet: For microarray analysis, 3 mg of lysate were incubated (1 h, 4°C) with 100 ml of a 50% (v/v) suspension of protein-A Sepharose beads precoated with 20 mg each of mouse anti-nucleolin or mouse IgG (Santa Cruz).

    Techniques: Quantitative RT-PCR, Standard Deviation

    Nucleolin promotes mRNA translation. ( A ) Forty-eight hours after transfection of HeLa cells with either Ctrl siRNA or NCL siRNA, lysates were prepared and fractionated through sucrose gradients into 12 gradient fractions; 40S and 60S, small and large ribosome subunits, respectively; 80S, monosome; LMWP and HMWP, low- and high-molecular weight polysomes, respectively (‘Materials and Methods’ section). ( B ) The relative distribution of nucleolin target mRNAs and housekeeping GAPDH mRNA was studied by RT–qPCR analysis of RNA in each of fraction. ( C ) De novo translation of proteins Usf2, Akt1, and Flot1 and housekeeping protein GAPDH ( left ) was determined in HeLa cells 48 h after transfection with the small RNAs shown; cells were incubated for 20 min with 35 S-labeled amino acids, whereupon the incorporation of the radiolabel into each protein was compared with the unchanged incorporation into GAPDH and with the nonspecific label incorporation detected in the IgG IP samples ( right ); details in Materials and Methods. Data are representative of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs

    doi: 10.1093/nar/gkr488

    Figure Lengend Snippet: Nucleolin promotes mRNA translation. ( A ) Forty-eight hours after transfection of HeLa cells with either Ctrl siRNA or NCL siRNA, lysates were prepared and fractionated through sucrose gradients into 12 gradient fractions; 40S and 60S, small and large ribosome subunits, respectively; 80S, monosome; LMWP and HMWP, low- and high-molecular weight polysomes, respectively (‘Materials and Methods’ section). ( B ) The relative distribution of nucleolin target mRNAs and housekeeping GAPDH mRNA was studied by RT–qPCR analysis of RNA in each of fraction. ( C ) De novo translation of proteins Usf2, Akt1, and Flot1 and housekeeping protein GAPDH ( left ) was determined in HeLa cells 48 h after transfection with the small RNAs shown; cells were incubated for 20 min with 35 S-labeled amino acids, whereupon the incorporation of the radiolabel into each protein was compared with the unchanged incorporation into GAPDH and with the nonspecific label incorporation detected in the IgG IP samples ( right ); details in Materials and Methods. Data are representative of three independent experiments.

    Article Snippet: For microarray analysis, 3 mg of lysate were incubated (1 h, 4°C) with 100 ml of a 50% (v/v) suspension of protein-A Sepharose beads precoated with 20 mg each of mouse anti-nucleolin or mouse IgG (Santa Cruz).

    Techniques: Transfection, Molecular Weight, Quantitative RT-PCR, Incubation, Labeling

    Induction of anti-HA humoral and cellular responses in mice vaccinated with VLP-HA/VSP-G. a – e Mice were orally vaccinated with VLPs and the humoral response against HA in different biological samples was evaluated. Levels of total serum anti-HA IgG ( a ), IgG1 and IgG2a ( b ), n = 8 from two independent experiments. Levels of IgA anti-HA in fecal extracts and BAL, n = 6 from two independent experiments ( c ). Levels of serum IgG anti-HA measured at different times post immunization, n = 6 from two independent experiments ( d ). Levels of serum IgG anti-HA in animals immunized with a mixture of VLP-HA plus VLP-VSP-G, n = 6 from two independent experiments ( e ). f Levels of serum IgG and fecal IgA in mice vaccinated with VLPs via oral or subcutaneous (s.c.) route, n = 8 from two independent experiments. g Mice were orally immunized with four weekly doses of 50 μg of recombinant ΔVSP1267 or vehicle. One week after the last immunization the presence of anti-VSP1267 antibodies in blood and fecal samples was checked, then these animals were orally immunized with VLP-HA/VSP-G, according to the protocol previously used. One week after the last VLP dose, the levels of serum IgG and fecal IgA anti-HA Abs were measured, n = 6 from two independent experiments. h , i Mice were orally vaccinated with VLPs and the cellular response against HA was evaluated. The frequency of HA-specific IFN-γ-secreting T cells (spot-forming colonies, SFC) was determined after antigen-specific re-stimulation of splenocytes, n = 7 from two independent experiments ( h ). Cytokines were measured in splenocyte supernatants and only those cytokines with detectable levels are shown, n = 6 from two independent experiments ( i ). Data were analyzed by one-way ANOVA and Tukey’s multiple comparison test ( a – f , h , i ) or by two-way ANOVA and Bonferroni post-tests ( g ). Values represent mean ± s.e.m. * p

    Journal: Nature Communications

    Article Title: Efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins

    doi: 10.1038/s41467-018-08265-9

    Figure Lengend Snippet: Induction of anti-HA humoral and cellular responses in mice vaccinated with VLP-HA/VSP-G. a – e Mice were orally vaccinated with VLPs and the humoral response against HA in different biological samples was evaluated. Levels of total serum anti-HA IgG ( a ), IgG1 and IgG2a ( b ), n = 8 from two independent experiments. Levels of IgA anti-HA in fecal extracts and BAL, n = 6 from two independent experiments ( c ). Levels of serum IgG anti-HA measured at different times post immunization, n = 6 from two independent experiments ( d ). Levels of serum IgG anti-HA in animals immunized with a mixture of VLP-HA plus VLP-VSP-G, n = 6 from two independent experiments ( e ). f Levels of serum IgG and fecal IgA in mice vaccinated with VLPs via oral or subcutaneous (s.c.) route, n = 8 from two independent experiments. g Mice were orally immunized with four weekly doses of 50 μg of recombinant ΔVSP1267 or vehicle. One week after the last immunization the presence of anti-VSP1267 antibodies in blood and fecal samples was checked, then these animals were orally immunized with VLP-HA/VSP-G, according to the protocol previously used. One week after the last VLP dose, the levels of serum IgG and fecal IgA anti-HA Abs were measured, n = 6 from two independent experiments. h , i Mice were orally vaccinated with VLPs and the cellular response against HA was evaluated. The frequency of HA-specific IFN-γ-secreting T cells (spot-forming colonies, SFC) was determined after antigen-specific re-stimulation of splenocytes, n = 7 from two independent experiments ( h ). Cytokines were measured in splenocyte supernatants and only those cytokines with detectable levels are shown, n = 6 from two independent experiments ( i ). Data were analyzed by one-way ANOVA and Tukey’s multiple comparison test ( a – f , h , i ) or by two-way ANOVA and Bonferroni post-tests ( g ). Values represent mean ± s.e.m. * p

    Article Snippet: The following horseradish peroxidase-conjugated antibodies were used: goat anti-mouse IgG1 (SouthernBiotech, Cat. # 1070-05, dilution 1/2000); goat anti-mouse IgG2 (SouthernBiotech, Cat. # 1080-05, dilution 1/2000); goat anti-mouse IgG (Molecular Probes, Cat. # G-21040, dilution 1/2000).

    Techniques: Mouse Assay, Recombinant

    Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP IgG1 in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test

    Journal: Nature Communications

    Article Title: PRMT5 is essential for B cell development and germinal center dynamics

    doi: 10.1038/s41467-018-07884-6

    Figure Lengend Snippet: Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP IgG1 in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test

    Article Snippet: Plates with cells were incubated in a humid chamber 12 h at 37 °C, 5% CO2 , then washed 6× with PBS 0.01% Tween-20, followed by incubation with goat anti-mouse IgG1-HRP (A10551, Life Technologies, 1/2000) diluted in culture media for 2 h at RT.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Flow Cytometry, Cytometry, Infection, Staining, Two Tailed Test