hrp horse radish peroxidase conjugated goat anti human  (Jackson Immuno)

 
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    Structured Review

    Jackson Immuno hrp horse radish peroxidase conjugated goat anti human
    Reactivity of human antibodies with pgp3 and control fusion proteins on Western blot . (A) GST fusion proteins as listed on top of the figure were analyzed in SDS gel (panel a) and parallel gels were blotted onto nitrocellulose membrane for reacting with the pooled positive human antiserum at various dilutions as listed along the left of the figure. The primary antibody reactivity was visualized with a goat anti-human <t>IgG</t> conjugated with <t>HRP</t> in ECL as described in the method section. The corresponding protein bands were indicated on the right of the figure. Note that the pooled human antiserum only minimally reacted with the pgp3 band at 1:4,000 (panel b) while remaining reactive with CPAF even after 1:1,000,000 dilution (panel f), suggesting that the human anti-pgp3 antibodies are highly conformation-dependent.
    Hrp Horse Radish Peroxidase Conjugated Goat Anti Human, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 85/100, based on 938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 938 article reviews
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    hrp horse radish peroxidase conjugated goat anti human - by Bioz Stars, 2020-08
    85/100 stars

    Images

    1) Product Images from "Antibodies from women urogenitally infected with C. trachomatis predominantly recognized the plasmid protein pgp3 in a conformation-dependent manner"

    Article Title: Antibodies from women urogenitally infected with C. trachomatis predominantly recognized the plasmid protein pgp3 in a conformation-dependent manner

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-90

    Reactivity of human antibodies with pgp3 and control fusion proteins on Western blot . (A) GST fusion proteins as listed on top of the figure were analyzed in SDS gel (panel a) and parallel gels were blotted onto nitrocellulose membrane for reacting with the pooled positive human antiserum at various dilutions as listed along the left of the figure. The primary antibody reactivity was visualized with a goat anti-human IgG conjugated with HRP in ECL as described in the method section. The corresponding protein bands were indicated on the right of the figure. Note that the pooled human antiserum only minimally reacted with the pgp3 band at 1:4,000 (panel b) while remaining reactive with CPAF even after 1:1,000,000 dilution (panel f), suggesting that the human anti-pgp3 antibodies are highly conformation-dependent.
    Figure Legend Snippet: Reactivity of human antibodies with pgp3 and control fusion proteins on Western blot . (A) GST fusion proteins as listed on top of the figure were analyzed in SDS gel (panel a) and parallel gels were blotted onto nitrocellulose membrane for reacting with the pooled positive human antiserum at various dilutions as listed along the left of the figure. The primary antibody reactivity was visualized with a goat anti-human IgG conjugated with HRP in ECL as described in the method section. The corresponding protein bands were indicated on the right of the figure. Note that the pooled human antiserum only minimally reacted with the pgp3 band at 1:4,000 (panel b) while remaining reactive with CPAF even after 1:1,000,000 dilution (panel f), suggesting that the human anti-pgp3 antibodies are highly conformation-dependent.

    Techniques Used: Western Blot, SDS-Gel

    2) Product Images from "Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis"

    Article Title: Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx326

    Association of hnRNP C with polysomes increases from G1 to mitosis. ( A ) MS measurements of members of the hnRNP family. Bar plot represents the logarithmic ratio of normalized MS intensities for M over G1; heatmaps reflect the relative abundance of each protein during mitosis in either ribosome-associated (‘Ribosome’) or total proteome (‘Total’) samples. ( B ) Polysomes were extracted from G1 and M cytoplasmic lysates by ultracentrifugation through a sucrose cushion, and protein content of the lysates and pellets was monitored by immunoblotting using antibodies to hnRNP C, Poly-A binding protein (PABPC1) and Ribosomal protein L26 (RPL26). ( C ) Polysomes were extracted from G1 and M cells by ultracentrifugation through a sucrose gradient, and protein content of each fraction was monitored by immunoblotting using antibodies to Ribosomal protein P0 (RPLP0) and either hnRNP C (top panel) or SRSF1 (bottom panel). The membrane was simultaneously incubated with either pair of antibodies to allow a more direct comparison of protein amounts. 40S, small ribosomal subunit. ( D ) Polysomes were extracted as in (A) and hnRNP C-bound complexes were immunoprecipitated using antibodies to hnRNP C or IgG as control, followed by incubation with biotin-conjugated puromycin to label nascent polypeptide chains. 5% of polysome pellet and 100% of hnRNP C IP were then subjected to immunoblot analysis using anti-hnRNP C and anti-RPL26, as well as streptavidin-HRP.
    Figure Legend Snippet: Association of hnRNP C with polysomes increases from G1 to mitosis. ( A ) MS measurements of members of the hnRNP family. Bar plot represents the logarithmic ratio of normalized MS intensities for M over G1; heatmaps reflect the relative abundance of each protein during mitosis in either ribosome-associated (‘Ribosome’) or total proteome (‘Total’) samples. ( B ) Polysomes were extracted from G1 and M cytoplasmic lysates by ultracentrifugation through a sucrose cushion, and protein content of the lysates and pellets was monitored by immunoblotting using antibodies to hnRNP C, Poly-A binding protein (PABPC1) and Ribosomal protein L26 (RPL26). ( C ) Polysomes were extracted from G1 and M cells by ultracentrifugation through a sucrose gradient, and protein content of each fraction was monitored by immunoblotting using antibodies to Ribosomal protein P0 (RPLP0) and either hnRNP C (top panel) or SRSF1 (bottom panel). The membrane was simultaneously incubated with either pair of antibodies to allow a more direct comparison of protein amounts. 40S, small ribosomal subunit. ( D ) Polysomes were extracted as in (A) and hnRNP C-bound complexes were immunoprecipitated using antibodies to hnRNP C or IgG as control, followed by incubation with biotin-conjugated puromycin to label nascent polypeptide chains. 5% of polysome pellet and 100% of hnRNP C IP were then subjected to immunoblot analysis using anti-hnRNP C and anti-RPL26, as well as streptavidin-HRP.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation, Immunoprecipitation

    3) Product Images from "Type I Interferon Impairs Specific Antibody Responses Early during Establishment of LCMV Infection"

    Article Title: Type I Interferon Impairs Specific Antibody Responses Early during Establishment of LCMV Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2016.00564

    IFNAR blockade restores NP-specific IgG1 responses during LCMV infection . B6 mice (four per group) were treated with α-IFNAR Ab (red), isotype control Ab (hatched), or left untreated (black) and were infected the next day with LCMV Cl13, VSV (gray), or mock infected (white). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum. (A,B,E,F) IFNAR blockade was conducted every second day until d8. In another series of experiments, anti-IFNAR treatment was stopped after either one injection (d-1) (C,G) or 11 injections every third day (d30) (D,H) . (A) LCMV nucleoprotein-specific IgG titers, (B–D) NP-specific IgG1, (E) NP-specific IgG2c, (F–H) total IgG titers monitored by ELISA. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p
    Figure Legend Snippet: IFNAR blockade restores NP-specific IgG1 responses during LCMV infection . B6 mice (four per group) were treated with α-IFNAR Ab (red), isotype control Ab (hatched), or left untreated (black) and were infected the next day with LCMV Cl13, VSV (gray), or mock infected (white). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum. (A,B,E,F) IFNAR blockade was conducted every second day until d8. In another series of experiments, anti-IFNAR treatment was stopped after either one injection (d-1) (C,G) or 11 injections every third day (d30) (D,H) . (A) LCMV nucleoprotein-specific IgG titers, (B–D) NP-specific IgG1, (E) NP-specific IgG2c, (F–H) total IgG titers monitored by ELISA. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p

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

    B cell-specific disruption of IFNAR restores NP-specific Ab responses and accelerates the development of LCMV nAbs . (A–C) B6, J H T/IFNAR −/− , or J H T/B6 mice (four per group) were infected with LCMV Cl13 (black for B6; red for J H T/IFNAR −/− ), VSV (gray), or were mock infected (white). Plain bars represent B6 mice and checkered bars J H T/IFNAR −/− B cell bone marrow chimeric mice. (A) NP-specific IgG1 and (B) total IgG responses monitored using ELISA on d8 following infection/NP 53 -CGG immunization. (C) Splenic follicular structures visualized using immunofluorescent staining of CD19 (red), MOMA-1 (green), and DAPI (blue) on spleen sections obtained on d8 postinfection. (D) Neutralization assay showing accelerated nAb responses in J H T/IFNAR −/− chimeric mice upon infection with LCMV Cl13. (A–C) Representative of two independent experiments. (D) Compilation of three independent experiments (B6 mice, n = 21; J H T/IFNAR −/− mice, n = 25; J H T/B6 mice, n = 8). Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group for (A–C) and one-way ANOVA for (D) . * p
    Figure Legend Snippet: B cell-specific disruption of IFNAR restores NP-specific Ab responses and accelerates the development of LCMV nAbs . (A–C) B6, J H T/IFNAR −/− , or J H T/B6 mice (four per group) were infected with LCMV Cl13 (black for B6; red for J H T/IFNAR −/− ), VSV (gray), or were mock infected (white). Plain bars represent B6 mice and checkered bars J H T/IFNAR −/− B cell bone marrow chimeric mice. (A) NP-specific IgG1 and (B) total IgG responses monitored using ELISA on d8 following infection/NP 53 -CGG immunization. (C) Splenic follicular structures visualized using immunofluorescent staining of CD19 (red), MOMA-1 (green), and DAPI (blue) on spleen sections obtained on d8 postinfection. (D) Neutralization assay showing accelerated nAb responses in J H T/IFNAR −/− chimeric mice upon infection with LCMV Cl13. (A–C) Representative of two independent experiments. (D) Compilation of three independent experiments (B6 mice, n = 21; J H T/IFNAR −/− mice, n = 25; J H T/B6 mice, n = 8). Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group for (A–C) and one-way ANOVA for (D) . * p

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Staining, Neutralization

    LCMV infection disrupts the splenic follicular architecture while increasing GC B cell numbers and non-specific Ab responses . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum and TD B cell responses were analyzed on d8 after infection. (A) Immunofluorescence showing CD19 (red), MOMA-1 (green), and DAPI (blue) expression on spleen sections. (B) Total B cell numbers and proportions were enumerated by flow cytometry. (C) Number and proportion of splenic GC B cells. (D) Number of total and NP-specific IgG-secreting cells detected by ELISPOT. (E) Relative Ab secretion of ASCs calculated by ex vivo measurement of secreted Abs produced by 10 5 splenocytes. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p
    Figure Legend Snippet: LCMV infection disrupts the splenic follicular architecture while increasing GC B cell numbers and non-specific Ab responses . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum and TD B cell responses were analyzed on d8 after infection. (A) Immunofluorescence showing CD19 (red), MOMA-1 (green), and DAPI (blue) expression on spleen sections. (B) Total B cell numbers and proportions were enumerated by flow cytometry. (C) Number and proportion of splenic GC B cells. (D) Number of total and NP-specific IgG-secreting cells detected by ELISPOT. (E) Relative Ab secretion of ASCs calculated by ex vivo measurement of secreted Abs produced by 10 5 splenocytes. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p

    Techniques Used: Infection, Mouse Assay, Injection, Immunofluorescence, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Ex Vivo, Produced

    LCMV infection triggers the expansion of T FH cells and induces their expression of B cell-activating cytokines . B6 mice (four per group) were infected with LCMV Cl13 (black bars or lines), LCMV WE (hatched), VSV (gray bars or dotted lines), or mock infected (white bars or shaded area). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum and CD4 + T cells were analyzed on d8 after infection. (A) Total CD4 + T cell numbers (top panel) and proportions (bottom panel) determined by flow cytometry. (B) T FH cell numbers and proportions determined by flow cytometry. (C) Proportion of T FH cells expressing PD-1, ICOS, and CD40L and (D) B cells expressing PD-L1, ICOSL, and CD40 determined by flow cytometry. (E) Serum BAFF concentration (left panel) and BAFF mRNA expression in total splenic CD4 T cells (right panel) measured using ELISA and qRT-PCR, respectively. (F) Intracellular cytokine levels (left panel) and mRNA expression (right panel) of IL-4 and IL-21 in CD4 T cells measured using flow cytometry and qRT-PCR, respectively. (G) NP-specific IgG1 response (left panel) and total IgG response (right panel) monitored upon infection with LCMV WE (hatched bars), VSV (gray), or mock-infected (white) infection along with injection of PD1-blocking Ab (red hatched or checkered bars). Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group and between non-treated and anti-PD1 Ab-treated LCMV-infected groups when indicated with brackets. * p
    Figure Legend Snippet: LCMV infection triggers the expansion of T FH cells and induces their expression of B cell-activating cytokines . B6 mice (four per group) were infected with LCMV Cl13 (black bars or lines), LCMV WE (hatched), VSV (gray bars or dotted lines), or mock infected (white bars or shaded area). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum and CD4 + T cells were analyzed on d8 after infection. (A) Total CD4 + T cell numbers (top panel) and proportions (bottom panel) determined by flow cytometry. (B) T FH cell numbers and proportions determined by flow cytometry. (C) Proportion of T FH cells expressing PD-1, ICOS, and CD40L and (D) B cells expressing PD-L1, ICOSL, and CD40 determined by flow cytometry. (E) Serum BAFF concentration (left panel) and BAFF mRNA expression in total splenic CD4 T cells (right panel) measured using ELISA and qRT-PCR, respectively. (F) Intracellular cytokine levels (left panel) and mRNA expression (right panel) of IL-4 and IL-21 in CD4 T cells measured using flow cytometry and qRT-PCR, respectively. (G) NP-specific IgG1 response (left panel) and total IgG response (right panel) monitored upon infection with LCMV WE (hatched bars), VSV (gray), or mock-infected (white) infection along with injection of PD1-blocking Ab (red hatched or checkered bars). Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group and between non-treated and anti-PD1 Ab-treated LCMV-infected groups when indicated with brackets. * p

    Techniques Used: Infection, Expressing, Mouse Assay, Injection, Flow Cytometry, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Blocking Assay

    IFNAR blockade partially restores TD B cell responses and splenic follicular structures . B6 mice (four per group) were treated with α-IFNAR Ab (checkered) or isotype control Ab (black) and were infected the next day with LCMV Cl13, VSV (gray), or mock infected (white). Mice were immunized the day of the infection with an i.p. injection of NP 53 -CGG in alum. IFNAR blockade was conducted every second day until analysis on d8. (A) Number of total (left panel) and NP-specific IgG-secreting (right panel) cells detected by ELISPOT. (B) Proportion of CD4 T cells among total lymphocytes and (C) total numbers (left panel) and proportions (right panel) of T FH cells among total CD4 T cells as determined by flow cytometry. (D) Intracellular IL-4 and IL-21 levels in T FH cells and (E) proportion of splenic GC B cells determined by flow cytometry. (F) Secretory capacity of ASCs as determined by measuring the quantity of secreted Abs produced by 10 5 splenocytes. (G) Proportion of B cells expressing PD-L1, ICOSL, and CD40. (H) Proportion of T FH cells expressing PD-1, ICOS, and CD40L. (I) Splenic follicular structures visualized using immunofluorescent staining of CD19 (red), MOMA-1 (green), and DAPI (blue) in tissue sections. (J) Proportion of CXCR4 + B cells among total (left panel) and GC B cells (right panel) determined by flow cytometry. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group and between isotype and IFNAR blocking Ab-treated groups when indicated with brackets. * p
    Figure Legend Snippet: IFNAR blockade partially restores TD B cell responses and splenic follicular structures . B6 mice (four per group) were treated with α-IFNAR Ab (checkered) or isotype control Ab (black) and were infected the next day with LCMV Cl13, VSV (gray), or mock infected (white). Mice were immunized the day of the infection with an i.p. injection of NP 53 -CGG in alum. IFNAR blockade was conducted every second day until analysis on d8. (A) Number of total (left panel) and NP-specific IgG-secreting (right panel) cells detected by ELISPOT. (B) Proportion of CD4 T cells among total lymphocytes and (C) total numbers (left panel) and proportions (right panel) of T FH cells among total CD4 T cells as determined by flow cytometry. (D) Intracellular IL-4 and IL-21 levels in T FH cells and (E) proportion of splenic GC B cells determined by flow cytometry. (F) Secretory capacity of ASCs as determined by measuring the quantity of secreted Abs produced by 10 5 splenocytes. (G) Proportion of B cells expressing PD-L1, ICOSL, and CD40. (H) Proportion of T FH cells expressing PD-1, ICOS, and CD40L. (I) Splenic follicular structures visualized using immunofluorescent staining of CD19 (red), MOMA-1 (green), and DAPI (blue) in tissue sections. (J) Proportion of CXCR4 + B cells among total (left panel) and GC B cells (right panel) determined by flow cytometry. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group and between isotype and IFNAR blocking Ab-treated groups when indicated with brackets. * p

    Techniques Used: Mouse Assay, Infection, Injection, Enzyme-linked Immunospot, Flow Cytometry, Cytometry, Produced, Expressing, Staining, Blocking Assay

    LCMV infection impairs the NP-specific Ab response in a T cell-dependent manner . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day (except when indicated otherwise) with an i.p. injection of NP 53 -CGG in alum (A,B,E,F) or NP 40 -FICOLL in PBS (C,D) . (A) NP-specific IgG1 response monitored by ELISA following NP 53 -CGG immunization. (B) Mice were infected as above and immunized with NP 53 -CGG the same day (d0) or 30 days after infection (d30), and IgG1 NP-specific responses were monitored by ELISA on d8 postimmunization. NP-specific IgG3 (C) or IgM (D) responses monitored by ELISA following NP 40 -FICOLL immunization. (E) ELISA plates were coated with NP 4 -BSA or NP 26 -BSA and high affinity Ab responses were measured as a ratio of Abs binding to NP 4 -BSA versus the total anti-NP IgG1 response binding to NP 26 -BSA. (F) Total serum IgG responses following concomitant infection and NP 53 -CGG immunization. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p
    Figure Legend Snippet: LCMV infection impairs the NP-specific Ab response in a T cell-dependent manner . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day (except when indicated otherwise) with an i.p. injection of NP 53 -CGG in alum (A,B,E,F) or NP 40 -FICOLL in PBS (C,D) . (A) NP-specific IgG1 response monitored by ELISA following NP 53 -CGG immunization. (B) Mice were infected as above and immunized with NP 53 -CGG the same day (d0) or 30 days after infection (d30), and IgG1 NP-specific responses were monitored by ELISA on d8 postimmunization. NP-specific IgG3 (C) or IgM (D) responses monitored by ELISA following NP 40 -FICOLL immunization. (E) ELISA plates were coated with NP 4 -BSA or NP 26 -BSA and high affinity Ab responses were measured as a ratio of Abs binding to NP 4 -BSA versus the total anti-NP IgG1 response binding to NP 26 -BSA. (F) Total serum IgG responses following concomitant infection and NP 53 -CGG immunization. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p

    Techniques Used: Infection, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay

    4) Product Images from "Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model"

    Article Title: Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.06.030

    (A) High anti-Tn antibody responses are induced by immunization with Qβ-Tn. The primary antibody response is IgG due to significantly higher IgG titers than IgM. The IgG titers from Qβ-Tn are much higher than those from control mice receiving Qβ only. (B) Titers of IgG subtypes antibodies (IgG1, IgG2b, IgG2c, and IgG3) elicited by Qβ-Tn immunization. All major IgG subtypes are induced suggesting balanced IgG responses.
    Figure Legend Snippet: (A) High anti-Tn antibody responses are induced by immunization with Qβ-Tn. The primary antibody response is IgG due to significantly higher IgG titers than IgM. The IgG titers from Qβ-Tn are much higher than those from control mice receiving Qβ only. (B) Titers of IgG subtypes antibodies (IgG1, IgG2b, IgG2c, and IgG3) elicited by Qβ-Tn immunization. All major IgG subtypes are induced suggesting balanced IgG responses.

    Techniques Used: Mouse Assay

    Recognition of (A) TA3Ha cells and (B) Jurkat cells by IgG antibodies in serum from mice before and after immunization with Qβ-Tn (for clarity, only one representative example of binding by postimmune serum is shown). Adapted with permission from Yin, Z., Wright, W. S., McKay, C., Baniel, C., Kaczanowska, K., Bentley, P., et al. (2015). Significant impact of immunogen design on the diversity of antibodies generated by carbohydrate-based anti-cancer vaccine. ACS Chemical Biology , 10 , 2364 – 2372 . Copyright 2015 American Chemical Society.
    Figure Legend Snippet: Recognition of (A) TA3Ha cells and (B) Jurkat cells by IgG antibodies in serum from mice before and after immunization with Qβ-Tn (for clarity, only one representative example of binding by postimmune serum is shown). Adapted with permission from Yin, Z., Wright, W. S., McKay, C., Baniel, C., Kaczanowska, K., Bentley, P., et al. (2015). Significant impact of immunogen design on the diversity of antibodies generated by carbohydrate-based anti-cancer vaccine. ACS Chemical Biology , 10 , 2364 – 2372 . Copyright 2015 American Chemical Society.

    Techniques Used: Mouse Assay, Binding Assay, Generated

    5) Product Images from "Cowpea Mosaic Virus Capsid, a Promising Carrier for the Development of Carbohydrate Based Anti-tumor Vaccines"

    Article Title: Cowpea Mosaic Virus Capsid, a Promising Carrier for the Development of Carbohydrate Based Anti-tumor Vaccines

    Journal: Chemistry (Weinheim an der Bergstrasse, Germany)

    doi: 10.1002/chem.200

    a) ELISA titers of anti-Tn IgG+IgM antibody in mouse sera prior to immunization (day 0), after immunization with Tn-1-S-CPMV (day 35) in the absence and presence of 0.1 M GalNAc. b) ELISA titers of anti-Tn IgM and IgG antibody in mouse sera after immunization
    Figure Legend Snippet: a) ELISA titers of anti-Tn IgG+IgM antibody in mouse sera prior to immunization (day 0), after immunization with Tn-1-S-CPMV (day 35) in the absence and presence of 0.1 M GalNAc. b) ELISA titers of anti-Tn IgM and IgG antibody in mouse sera after immunization

    Techniques Used: Enzyme-linked Immunosorbent Assay

    6) Product Images from "Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model"

    Article Title: Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.06.030

    (A) High anti-Tn antibody responses are induced by immunization with Qβ-Tn. The primary antibody response is IgG due to significantly higher IgG titers than IgM. The IgG titers from Qβ-Tn are much higher than those from control mice receiving Qβ only. (B) Titers of IgG subtypes antibodies (IgG1, IgG2b, IgG2c, and IgG3) elicited by Qβ-Tn immunization. All major IgG subtypes are induced suggesting balanced IgG responses.
    Figure Legend Snippet: (A) High anti-Tn antibody responses are induced by immunization with Qβ-Tn. The primary antibody response is IgG due to significantly higher IgG titers than IgM. The IgG titers from Qβ-Tn are much higher than those from control mice receiving Qβ only. (B) Titers of IgG subtypes antibodies (IgG1, IgG2b, IgG2c, and IgG3) elicited by Qβ-Tn immunization. All major IgG subtypes are induced suggesting balanced IgG responses.

    Techniques Used: Mouse Assay

    Recognition of (A) TA3Ha cells and (B) Jurkat cells by IgG antibodies in serum from mice before and after immunization with Qβ-Tn (for clarity, only one representative example of binding by postimmune serum is shown). Adapted with permission from Yin, Z., Wright, W. S., McKay, C., Baniel, C., Kaczanowska, K., Bentley, P., et al. (2015). Significant impact of immunogen design on the diversity of antibodies generated by carbohydrate-based anti-cancer vaccine. ACS Chemical Biology , 10 , 2364 – 2372 . Copyright 2015 American Chemical Society.
    Figure Legend Snippet: Recognition of (A) TA3Ha cells and (B) Jurkat cells by IgG antibodies in serum from mice before and after immunization with Qβ-Tn (for clarity, only one representative example of binding by postimmune serum is shown). Adapted with permission from Yin, Z., Wright, W. S., McKay, C., Baniel, C., Kaczanowska, K., Bentley, P., et al. (2015). Significant impact of immunogen design on the diversity of antibodies generated by carbohydrate-based anti-cancer vaccine. ACS Chemical Biology , 10 , 2364 – 2372 . Copyright 2015 American Chemical Society.

    Techniques Used: Mouse Assay, Binding Assay, Generated

    7) Product Images from "Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections"

    Article Title: Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0166621

    Immunization of mice with the live-attenuated double mutant (Δ720Δ hemB ) induces a strong humoral response against S . aureus bovine mastitis isolates and against a specific cell-wall associated antigen (IsdH). Mice were immunized as previously described: serums were collected before priming immunization (preimmune, open circles) and ten days after the boost immunization (immune, blue squares). A. IgG titers rise with increasing immunization doses (10 6 , 10 7 , 10 8 CFU) of the live-attenuated mutant Δ vraG Δ hemB : each dot represents the total IgG titer of one mouse against a Δ vraG Δ hemB whole cell extract. Medians are represented by thick lines for immune titers and dashed lines for preimmune titers. Titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey’s multiple comparisons test: ****: P ≤ 0.0001). B. Immunization with the live-attenuated mutant Δ vraG Δ hemB confers high IgG titers against components that are shared by mastitis strains of commonly found spa types. Each dot represents the total IgG titer of one mouse against the whole cell extract of the indicated strain. Medians are represented by thick lines for immune titers and dashed lines for preimmune titers. All immune titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey’s multiple comparisons test: P ≤ 0.0001 for all groups). C. Immunization with the live-attenuated mutant Δ vraG Δ hemB confers specific IgG titers against the cell-wall associated protein IsdH. Each dot represents the total IgG titer of one mouse against recombinant IsdH. Compared groups were immunized with the 10 8 CFU of the live-attenuated Δ vraG Δ hemB (ΔΔ) or 25 μg of the purified recombinant IsdH protein (IsdH). D. IgG isotype ratios (IgG2a/IgG1) of mice immunized with the live-attenuated mutant Δ vraG Δ hemB (open diamonds) or immunized with the recombinant IsdH (black diamonds), against whole-cell extracts of strain Δ vraG Δ hemB (vs ΔΔ) or against the recombinant IsdH protein (vs IsdH). Each diamond represents the IgG2a/IgG1 titer ratio for one mouse. Medians are represented by thick lines (One-way ANOVA and Dunn’s multiple comparison test: *: P ≤ 0.05).
    Figure Legend Snippet: Immunization of mice with the live-attenuated double mutant (Δ720Δ hemB ) induces a strong humoral response against S . aureus bovine mastitis isolates and against a specific cell-wall associated antigen (IsdH). Mice were immunized as previously described: serums were collected before priming immunization (preimmune, open circles) and ten days after the boost immunization (immune, blue squares). A. IgG titers rise with increasing immunization doses (10 6 , 10 7 , 10 8 CFU) of the live-attenuated mutant Δ vraG Δ hemB : each dot represents the total IgG titer of one mouse against a Δ vraG Δ hemB whole cell extract. Medians are represented by thick lines for immune titers and dashed lines for preimmune titers. Titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey’s multiple comparisons test: ****: P ≤ 0.0001). B. Immunization with the live-attenuated mutant Δ vraG Δ hemB confers high IgG titers against components that are shared by mastitis strains of commonly found spa types. Each dot represents the total IgG titer of one mouse against the whole cell extract of the indicated strain. Medians are represented by thick lines for immune titers and dashed lines for preimmune titers. All immune titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey’s multiple comparisons test: P ≤ 0.0001 for all groups). C. Immunization with the live-attenuated mutant Δ vraG Δ hemB confers specific IgG titers against the cell-wall associated protein IsdH. Each dot represents the total IgG titer of one mouse against recombinant IsdH. Compared groups were immunized with the 10 8 CFU of the live-attenuated Δ vraG Δ hemB (ΔΔ) or 25 μg of the purified recombinant IsdH protein (IsdH). D. IgG isotype ratios (IgG2a/IgG1) of mice immunized with the live-attenuated mutant Δ vraG Δ hemB (open diamonds) or immunized with the recombinant IsdH (black diamonds), against whole-cell extracts of strain Δ vraG Δ hemB (vs ΔΔ) or against the recombinant IsdH protein (vs IsdH). Each diamond represents the IgG2a/IgG1 titer ratio for one mouse. Medians are represented by thick lines (One-way ANOVA and Dunn’s multiple comparison test: *: P ≤ 0.05).

    Techniques Used: Mouse Assay, Mutagenesis, Recombinant, Purification

    8) Product Images from "A Quantitative High-Throughput In Vitro Splicing Assay Identifies Inhibitors of Spliceosome Catalysis"

    Article Title: A Quantitative High-Throughput In Vitro Splicing Assay Identifies Inhibitors of Spliceosome Catalysis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.05788-11

    A high-throughput assay (EJIPT) utilizing EJC binding to spliced mRNAs to measure splicing in vitro . Splicing reaction mixtures containing biotin-labeled pre-mRNA, 20 μM each compound, splicing extract, and 0.5 mM ATP are assembled by liquid handler in 384-well plates and incubated for 1.5 h at 30°C. Reaction mixtures are then transferred to a NeutrAvidin-coated plate, which captures biotin-labeled RNAs/RNPs postsplicing. Anti-eIF4AIII (3F1) antibody is added to detect splicing-dependent EJC formation on spliced mRNAs. Following a washing step, incubation with an HRP-conjugated anti-mouse IgG secondary antibody, and then addition of chemiluminescent substrate, signals representing eIF4AIII/mRNA complexes are obtained by an automatic plate reader.
    Figure Legend Snippet: A high-throughput assay (EJIPT) utilizing EJC binding to spliced mRNAs to measure splicing in vitro . Splicing reaction mixtures containing biotin-labeled pre-mRNA, 20 μM each compound, splicing extract, and 0.5 mM ATP are assembled by liquid handler in 384-well plates and incubated for 1.5 h at 30°C. Reaction mixtures are then transferred to a NeutrAvidin-coated plate, which captures biotin-labeled RNAs/RNPs postsplicing. Anti-eIF4AIII (3F1) antibody is added to detect splicing-dependent EJC formation on spliced mRNAs. Following a washing step, incubation with an HRP-conjugated anti-mouse IgG secondary antibody, and then addition of chemiluminescent substrate, signals representing eIF4AIII/mRNA complexes are obtained by an automatic plate reader.

    Techniques Used: High Throughput Screening Assay, Binding Assay, In Vitro, Labeling, Incubation

    9) Product Images from "Type I Interferon Impairs Specific Antibody Responses Early during Establishment of LCMV Infection"

    Article Title: Type I Interferon Impairs Specific Antibody Responses Early during Establishment of LCMV Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2016.00564

    IFNAR blockade restores NP-specific IgG1 responses during LCMV infection . B6 mice (four per group) were treated with α-IFNAR Ab (red), isotype control Ab (hatched), or left untreated (black) and were infected the next day with LCMV Cl13, VSV (gray), or mock infected (white). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum. (A,B,E,F) IFNAR blockade was conducted every second day until d8. In another series of experiments, anti-IFNAR treatment was stopped after either one injection (d-1) (C,G) or 11 injections every third day (d30) (D,H) . (A) LCMV nucleoprotein-specific IgG titers, (B–D) NP-specific IgG1, (E) NP-specific IgG2c, (F–H) total IgG titers monitored by ELISA. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p
    Figure Legend Snippet: IFNAR blockade restores NP-specific IgG1 responses during LCMV infection . B6 mice (four per group) were treated with α-IFNAR Ab (red), isotype control Ab (hatched), or left untreated (black) and were infected the next day with LCMV Cl13, VSV (gray), or mock infected (white). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum. (A,B,E,F) IFNAR blockade was conducted every second day until d8. In another series of experiments, anti-IFNAR treatment was stopped after either one injection (d-1) (C,G) or 11 injections every third day (d30) (D,H) . (A) LCMV nucleoprotein-specific IgG titers, (B–D) NP-specific IgG1, (E) NP-specific IgG2c, (F–H) total IgG titers monitored by ELISA. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p

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

    B cell-specific disruption of IFNAR restores NP-specific Ab responses and accelerates the development of LCMV nAbs . (A–C) B6, J H T/IFNAR −/− , or J H T/B6 mice (four per group) were infected with LCMV Cl13 (black for B6; red for J H T/IFNAR −/− ), VSV (gray), or were mock infected (white). Plain bars represent B6 mice and checkered bars J H T/IFNAR −/− B cell bone marrow chimeric mice. (A) NP-specific IgG1 and (B) total IgG responses monitored using ELISA on d8 following infection/NP 53 -CGG immunization. (C) Splenic follicular structures visualized using immunofluorescent staining of CD19 (red), MOMA-1 (green), and DAPI (blue) on spleen sections obtained on d8 postinfection. (D) Neutralization assay showing accelerated nAb responses in J H T/IFNAR −/− chimeric mice upon infection with LCMV Cl13. (A–C) Representative of two independent experiments. (D) Compilation of three independent experiments (B6 mice, n = 21; J H T/IFNAR −/− mice, n = 25; J H T/B6 mice, n = 8). Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group for (A–C) and one-way ANOVA for (D) . * p
    Figure Legend Snippet: B cell-specific disruption of IFNAR restores NP-specific Ab responses and accelerates the development of LCMV nAbs . (A–C) B6, J H T/IFNAR −/− , or J H T/B6 mice (four per group) were infected with LCMV Cl13 (black for B6; red for J H T/IFNAR −/− ), VSV (gray), or were mock infected (white). Plain bars represent B6 mice and checkered bars J H T/IFNAR −/− B cell bone marrow chimeric mice. (A) NP-specific IgG1 and (B) total IgG responses monitored using ELISA on d8 following infection/NP 53 -CGG immunization. (C) Splenic follicular structures visualized using immunofluorescent staining of CD19 (red), MOMA-1 (green), and DAPI (blue) on spleen sections obtained on d8 postinfection. (D) Neutralization assay showing accelerated nAb responses in J H T/IFNAR −/− chimeric mice upon infection with LCMV Cl13. (A–C) Representative of two independent experiments. (D) Compilation of three independent experiments (B6 mice, n = 21; J H T/IFNAR −/− mice, n = 25; J H T/B6 mice, n = 8). Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group for (A–C) and one-way ANOVA for (D) . * p

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Staining, Neutralization

    LCMV infection disrupts the splenic follicular architecture while increasing GC B cell numbers and non-specific Ab responses . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum and TD B cell responses were analyzed on d8 after infection. (A) Immunofluorescence showing CD19 (red), MOMA-1 (green), and DAPI (blue) expression on spleen sections. (B) Total B cell numbers and proportions were enumerated by flow cytometry. (C) Number and proportion of splenic GC B cells. (D) Number of total and NP-specific IgG-secreting cells detected by ELISPOT. (E) Relative Ab secretion of ASCs calculated by ex vivo measurement of secreted Abs produced by 10 5 splenocytes. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p
    Figure Legend Snippet: LCMV infection disrupts the splenic follicular architecture while increasing GC B cell numbers and non-specific Ab responses . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum and TD B cell responses were analyzed on d8 after infection. (A) Immunofluorescence showing CD19 (red), MOMA-1 (green), and DAPI (blue) expression on spleen sections. (B) Total B cell numbers and proportions were enumerated by flow cytometry. (C) Number and proportion of splenic GC B cells. (D) Number of total and NP-specific IgG-secreting cells detected by ELISPOT. (E) Relative Ab secretion of ASCs calculated by ex vivo measurement of secreted Abs produced by 10 5 splenocytes. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p

    Techniques Used: Infection, Mouse Assay, Injection, Immunofluorescence, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Ex Vivo, Produced

    LCMV infection triggers the expansion of T FH cells and induces their expression of B cell-activating cytokines . B6 mice (four per group) were infected with LCMV Cl13 (black bars or lines), LCMV WE (hatched), VSV (gray bars or dotted lines), or mock infected (white bars or shaded area). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum and CD4 + T cells were analyzed on d8 after infection. (A) Total CD4 + T cell numbers (top panel) and proportions (bottom panel) determined by flow cytometry. (B) T FH cell numbers and proportions determined by flow cytometry. (C) Proportion of T FH cells expressing PD-1, ICOS, and CD40L and (D) B cells expressing PD-L1, ICOSL, and CD40 determined by flow cytometry. (E) Serum BAFF concentration (left panel) and BAFF mRNA expression in total splenic CD4 T cells (right panel) measured using ELISA and qRT-PCR, respectively. (F) Intracellular cytokine levels (left panel) and mRNA expression (right panel) of IL-4 and IL-21 in CD4 T cells measured using flow cytometry and qRT-PCR, respectively. (G) NP-specific IgG1 response (left panel) and total IgG response (right panel) monitored upon infection with LCMV WE (hatched bars), VSV (gray), or mock-infected (white) infection along with injection of PD1-blocking Ab (red hatched or checkered bars). Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group and between non-treated and anti-PD1 Ab-treated LCMV-infected groups when indicated with brackets. * p
    Figure Legend Snippet: LCMV infection triggers the expansion of T FH cells and induces their expression of B cell-activating cytokines . B6 mice (four per group) were infected with LCMV Cl13 (black bars or lines), LCMV WE (hatched), VSV (gray bars or dotted lines), or mock infected (white bars or shaded area). Mice were immunized the same day with an i.p. injection of NP 53 -CGG in alum and CD4 + T cells were analyzed on d8 after infection. (A) Total CD4 + T cell numbers (top panel) and proportions (bottom panel) determined by flow cytometry. (B) T FH cell numbers and proportions determined by flow cytometry. (C) Proportion of T FH cells expressing PD-1, ICOS, and CD40L and (D) B cells expressing PD-L1, ICOSL, and CD40 determined by flow cytometry. (E) Serum BAFF concentration (left panel) and BAFF mRNA expression in total splenic CD4 T cells (right panel) measured using ELISA and qRT-PCR, respectively. (F) Intracellular cytokine levels (left panel) and mRNA expression (right panel) of IL-4 and IL-21 in CD4 T cells measured using flow cytometry and qRT-PCR, respectively. (G) NP-specific IgG1 response (left panel) and total IgG response (right panel) monitored upon infection with LCMV WE (hatched bars), VSV (gray), or mock-infected (white) infection along with injection of PD1-blocking Ab (red hatched or checkered bars). Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group and between non-treated and anti-PD1 Ab-treated LCMV-infected groups when indicated with brackets. * p

    Techniques Used: Infection, Expressing, Mouse Assay, Injection, Flow Cytometry, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Blocking Assay

    IFNAR blockade partially restores TD B cell responses and splenic follicular structures . B6 mice (four per group) were treated with α-IFNAR Ab (checkered) or isotype control Ab (black) and were infected the next day with LCMV Cl13, VSV (gray), or mock infected (white). Mice were immunized the day of the infection with an i.p. injection of NP 53 -CGG in alum. IFNAR blockade was conducted every second day until analysis on d8. (A) Number of total (left panel) and NP-specific IgG-secreting (right panel) cells detected by ELISPOT. (B) Proportion of CD4 T cells among total lymphocytes and (C) total numbers (left panel) and proportions (right panel) of T FH cells among total CD4 T cells as determined by flow cytometry. (D) Intracellular IL-4 and IL-21 levels in T FH cells and (E) proportion of splenic GC B cells determined by flow cytometry. (F) Secretory capacity of ASCs as determined by measuring the quantity of secreted Abs produced by 10 5 splenocytes. (G) Proportion of B cells expressing PD-L1, ICOSL, and CD40. (H) Proportion of T FH cells expressing PD-1, ICOS, and CD40L. (I) Splenic follicular structures visualized using immunofluorescent staining of CD19 (red), MOMA-1 (green), and DAPI (blue) in tissue sections. (J) Proportion of CXCR4 + B cells among total (left panel) and GC B cells (right panel) determined by flow cytometry. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group and between isotype and IFNAR blocking Ab-treated groups when indicated with brackets. * p
    Figure Legend Snippet: IFNAR blockade partially restores TD B cell responses and splenic follicular structures . B6 mice (four per group) were treated with α-IFNAR Ab (checkered) or isotype control Ab (black) and were infected the next day with LCMV Cl13, VSV (gray), or mock infected (white). Mice were immunized the day of the infection with an i.p. injection of NP 53 -CGG in alum. IFNAR blockade was conducted every second day until analysis on d8. (A) Number of total (left panel) and NP-specific IgG-secreting (right panel) cells detected by ELISPOT. (B) Proportion of CD4 T cells among total lymphocytes and (C) total numbers (left panel) and proportions (right panel) of T FH cells among total CD4 T cells as determined by flow cytometry. (D) Intracellular IL-4 and IL-21 levels in T FH cells and (E) proportion of splenic GC B cells determined by flow cytometry. (F) Secretory capacity of ASCs as determined by measuring the quantity of secreted Abs produced by 10 5 splenocytes. (G) Proportion of B cells expressing PD-L1, ICOSL, and CD40. (H) Proportion of T FH cells expressing PD-1, ICOS, and CD40L. (I) Splenic follicular structures visualized using immunofluorescent staining of CD19 (red), MOMA-1 (green), and DAPI (blue) in tissue sections. (J) Proportion of CXCR4 + B cells among total (left panel) and GC B cells (right panel) determined by flow cytometry. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group and between isotype and IFNAR blocking Ab-treated groups when indicated with brackets. * p

    Techniques Used: Mouse Assay, Infection, Injection, Enzyme-linked Immunospot, Flow Cytometry, Cytometry, Produced, Expressing, Staining, Blocking Assay

    LCMV infection impairs the NP-specific Ab response in a T cell-dependent manner . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day (except when indicated otherwise) with an i.p. injection of NP 53 -CGG in alum (A,B,E,F) or NP 40 -FICOLL in PBS (C,D) . (A) NP-specific IgG1 response monitored by ELISA following NP 53 -CGG immunization. (B) Mice were infected as above and immunized with NP 53 -CGG the same day (d0) or 30 days after infection (d30), and IgG1 NP-specific responses were monitored by ELISA on d8 postimmunization. NP-specific IgG3 (C) or IgM (D) responses monitored by ELISA following NP 40 -FICOLL immunization. (E) ELISA plates were coated with NP 4 -BSA or NP 26 -BSA and high affinity Ab responses were measured as a ratio of Abs binding to NP 4 -BSA versus the total anti-NP IgG1 response binding to NP 26 -BSA. (F) Total serum IgG responses following concomitant infection and NP 53 -CGG immunization. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p
    Figure Legend Snippet: LCMV infection impairs the NP-specific Ab response in a T cell-dependent manner . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day (except when indicated otherwise) with an i.p. injection of NP 53 -CGG in alum (A,B,E,F) or NP 40 -FICOLL in PBS (C,D) . (A) NP-specific IgG1 response monitored by ELISA following NP 53 -CGG immunization. (B) Mice were infected as above and immunized with NP 53 -CGG the same day (d0) or 30 days after infection (d30), and IgG1 NP-specific responses were monitored by ELISA on d8 postimmunization. NP-specific IgG3 (C) or IgM (D) responses monitored by ELISA following NP 40 -FICOLL immunization. (E) ELISA plates were coated with NP 4 -BSA or NP 26 -BSA and high affinity Ab responses were measured as a ratio of Abs binding to NP 4 -BSA versus the total anti-NP IgG1 response binding to NP 26 -BSA. (F) Total serum IgG responses following concomitant infection and NP 53 -CGG immunization. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p

    Techniques Used: Infection, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay

    10) Product Images from "Modification of the Primary Tumor Microenvironment by Transforming Growth Factor ?-Epidermal Growth Factor Receptor Signaling Promotes Metastasis in an Orthotopic Colon Cancer Model"

    Article Title: Modification of the Primary Tumor Microenvironment by Transforming Growth Factor ?-Epidermal Growth Factor Receptor Signaling Promotes Metastasis in an Orthotopic Colon Cancer Model

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2008.071147

    Immunohistochemical analyses of VEGFA, IL-8, MMP-2, MMP-9, and α-SMA expression in orthotopically implanted colon tumors. The microenvironment of TGFα-secreting tumors (KM12C parent, C9, and C10-TGFα was enriched in VEGFA, IL-8,
    Figure Legend Snippet: Immunohistochemical analyses of VEGFA, IL-8, MMP-2, MMP-9, and α-SMA expression in orthotopically implanted colon tumors. The microenvironment of TGFα-secreting tumors (KM12C parent, C9, and C10-TGFα was enriched in VEGFA, IL-8,

    Techniques Used: Immunohistochemistry, Expressing

    Immunohistochemical analyses of TGFα, EGFR, and phosphorylated EGFR expression on tumor cells and tumor-associated endothelial cells in orthotopically implanted colon tumors. The pattern of tumor TGFα expression in the mouse colon is identical
    Figure Legend Snippet: Immunohistochemical analyses of TGFα, EGFR, and phosphorylated EGFR expression on tumor cells and tumor-associated endothelial cells in orthotopically implanted colon tumors. The pattern of tumor TGFα expression in the mouse colon is identical

    Techniques Used: Immunohistochemistry, Expressing

    11) Product Images from "Calcium Channel Blocker Verapamil Enhances Endoplasmic Reticulum Stress and Cell Death Induced by Proteasome Inhibition in Myeloma Cells 1Calcium Channel Blocker Verapamil Enhances Endoplasmic Reticulum Stress and Cell Death Induced by Proteasome Inhibition in Myeloma Cells 1 2"

    Article Title: Calcium Channel Blocker Verapamil Enhances Endoplasmic Reticulum Stress and Cell Death Induced by Proteasome Inhibition in Myeloma Cells 1Calcium Channel Blocker Verapamil Enhances Endoplasmic Reticulum Stress and Cell Death Induced by Proteasome Inhibition in Myeloma Cells 1 2

    Journal: Neoplasia (New York, N.Y.)

    doi:

    JK-6L cells show enhanced protein accumulation in the presence of bortezomib and verapamil. (A) Western blot analysis of detergent-soluble and detergent-insoluble fractions of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hours. Immunoblot analysiswas done using an anti-ubiquitin antibody. To detect free ubiquitin, a denser gel was used. One representative immunoblot of two independent experiments is shown. (B) Western blot analysis of total cell lysates fromJK-6L cells treated with 10 nM bortezomib and/or 70 µMverapamil for 16 hours. Immunoblot analysis was done using HRP-conjugated anti-IgG antibody. One representative immunoblot of two independent experiments is shown. (C) IgG concentration in the supernatant of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hourswas determined by ELISA. The absorbance (OD)wasmeasured using a spectrophotometer. Mean values and SDwere calculated fromhexaplicates. Data represent one of two independently done experiments. Student's t -test for unpaired heteroscedastic samples was used for statistical analysis. # P
    Figure Legend Snippet: JK-6L cells show enhanced protein accumulation in the presence of bortezomib and verapamil. (A) Western blot analysis of detergent-soluble and detergent-insoluble fractions of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hours. Immunoblot analysiswas done using an anti-ubiquitin antibody. To detect free ubiquitin, a denser gel was used. One representative immunoblot of two independent experiments is shown. (B) Western blot analysis of total cell lysates fromJK-6L cells treated with 10 nM bortezomib and/or 70 µMverapamil for 16 hours. Immunoblot analysis was done using HRP-conjugated anti-IgG antibody. One representative immunoblot of two independent experiments is shown. (C) IgG concentration in the supernatant of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hourswas determined by ELISA. The absorbance (OD)wasmeasured using a spectrophotometer. Mean values and SDwere calculated fromhexaplicates. Data represent one of two independently done experiments. Student's t -test for unpaired heteroscedastic samples was used for statistical analysis. # P

    Techniques Used: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Spectrophotometry

    12) Product Images from "Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein"

    Article Title: Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

    Journal: Journal of Biomedical Research

    doi: 10.7555/JBR.30.20140157

    ELISA of the immunized rabbit sera. One hundred μL commercial EV71 VP1 protein (50 μg/L) was performed to coat ELISA plates. The immunized rabbit sera obtained from the previous experiments were diluted to 1:200. Biotinylated anti-rabbit IgG was diluted to 1:2000, HRP-conjugated streptavidin was diluted to 1:2000. The values represent the antibody response of 5 rabbits in the same group (average OD values with the associated SD).
    Figure Legend Snippet: ELISA of the immunized rabbit sera. One hundred μL commercial EV71 VP1 protein (50 μg/L) was performed to coat ELISA plates. The immunized rabbit sera obtained from the previous experiments were diluted to 1:200. Biotinylated anti-rabbit IgG was diluted to 1:2000, HRP-conjugated streptavidin was diluted to 1:2000. The values represent the antibody response of 5 rabbits in the same group (average OD values with the associated SD).

    Techniques Used: Enzyme-linked Immunosorbent Assay

    The expression and secretion of VP1 DNA vaccines. Mouse monoclonal VP1 antibody was used as the primary antibody (at a dilution of 1:1,000). HRP-conjugated goat anti-mouse IgG was diluted to 1:10,000. A: ELISA. Lysates (L, at a dilution of 1:10) and supernatants (S, at a dilution of 1:5) were used as antigens to coat the wells. The results represent the data from 3 biological replicates and are expressed as the mean ± SD (standard deviation). B: Western blotting assay: 20 μL original lysates or supernatants were loaded in each lane. The molecular weights of the proteins are as follows: wt-VP1 L ~35kD, tPA-VP1 L ~38kD, VP1-d L ~71kD and VP1-hFc L ~62kD. The molecular weights of the secreted proteins in the supernatant are greater than the corresponding intracellular forms due to protein glycosylation. To compare the protein expression levels of the different VP1 constructs and to determine if wt-VP1 and tPA-VP1 could be secreted, equal amounts of the 4 VP1 constructs were transfected into 293T cells. Lysates (L) and supernatants (S) of transfected cells were harvested and assayed using identical conditions. C: Comparison of the protein levels in the lysates. Twenty μL lysates were loaded in each lane. Anti-beta-actin monoclonal antibody was diluted 1:500. D: Detection of protein in the supernatants. Forty mL of supernatant from transfected cells was concentrated to 1 mL; 10 μL of the concentrated supernatants was analyzed by Western blotting.
    Figure Legend Snippet: The expression and secretion of VP1 DNA vaccines. Mouse monoclonal VP1 antibody was used as the primary antibody (at a dilution of 1:1,000). HRP-conjugated goat anti-mouse IgG was diluted to 1:10,000. A: ELISA. Lysates (L, at a dilution of 1:10) and supernatants (S, at a dilution of 1:5) were used as antigens to coat the wells. The results represent the data from 3 biological replicates and are expressed as the mean ± SD (standard deviation). B: Western blotting assay: 20 μL original lysates or supernatants were loaded in each lane. The molecular weights of the proteins are as follows: wt-VP1 L ~35kD, tPA-VP1 L ~38kD, VP1-d L ~71kD and VP1-hFc L ~62kD. The molecular weights of the secreted proteins in the supernatant are greater than the corresponding intracellular forms due to protein glycosylation. To compare the protein expression levels of the different VP1 constructs and to determine if wt-VP1 and tPA-VP1 could be secreted, equal amounts of the 4 VP1 constructs were transfected into 293T cells. Lysates (L) and supernatants (S) of transfected cells were harvested and assayed using identical conditions. C: Comparison of the protein levels in the lysates. Twenty μL lysates were loaded in each lane. Anti-beta-actin monoclonal antibody was diluted 1:500. D: Detection of protein in the supernatants. Forty mL of supernatant from transfected cells was concentrated to 1 mL; 10 μL of the concentrated supernatants was analyzed by Western blotting.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot, Construct, Transfection

    The immunogenicity of VP1 DNA vaccines in BALB/c mouse. One hundred μL EV71 VP1 protein (50 μg/L) was used to coat the wells to analyze the mouse sera for immunization (at a dilution of 1:200). HRP-conjugated goat anti-mouse IgG was diluted to 1:5,000. A: Temporal VP1-specific antibody responses in mouse immune sera. The arrows indicate the time points of DNA immunizations. Each curve represents the antibody response of 5 mice in the same group (average OD values with the associated SD). B: Peak level antibody titer in the mouse sera (average OD values with the associated SD). C: Western blotting assay of VP1 constructs. 20 μL of lysates (L) or supernatants (S) was added in each lane. Mouse sera immunized with different VP1 DNA vaccines were diluted to 1:200. HRP-conjugated goat anti-mouse IgG was diluted to 1:10,000.
    Figure Legend Snippet: The immunogenicity of VP1 DNA vaccines in BALB/c mouse. One hundred μL EV71 VP1 protein (50 μg/L) was used to coat the wells to analyze the mouse sera for immunization (at a dilution of 1:200). HRP-conjugated goat anti-mouse IgG was diluted to 1:5,000. A: Temporal VP1-specific antibody responses in mouse immune sera. The arrows indicate the time points of DNA immunizations. Each curve represents the antibody response of 5 mice in the same group (average OD values with the associated SD). B: Peak level antibody titer in the mouse sera (average OD values with the associated SD). C: Western blotting assay of VP1 constructs. 20 μL of lysates (L) or supernatants (S) was added in each lane. Mouse sera immunized with different VP1 DNA vaccines were diluted to 1:200. HRP-conjugated goat anti-mouse IgG was diluted to 1:10,000.

    Techniques Used: Mouse Assay, Western Blot, Construct

    Detection of purified VP1-hFc protein. Primary antibody used to detect VP1-hFc protein was wt-VP1-immunized rabbit serum (at a dilution of 1:500). HRP-conjugated goat anti-rabbit IgG was diluted to 1:10,000. A: SDS-PAGE analysis of purified VP1-hFc protein. All of the purified VP1-hFc proteins were mixed and concentrated to 1 mL for SDS-PAGE analysis. Different amounts of the protein samples (8 μL, 16 μL and 32 μL; initial concentration of 0.16 μg/μL) were detected. Different amounts of BSA (1 μg, 2 μg and 4 μg) were added as a semi-quantitative gauge. B: ELISA. P1 represents 1 μg of purified and concentrated VP1-hFc protein (0.16 μg/μL) in 100 μL of PBS. Then, P1 was serially diluted 2-fold (P2-P10). Vector-S and VP1-hFc-S represent the original supernatants diluted to 1:5. C: Western blotting assay. Lane 1 shows 20 μL of the original VP1-hFc-S; Lanes 2-7 indicate 40 μL, 20 μL, 10 μL, 5 μL, 2.5 μL and 1.25 μL of purified VP1-hFc protein, respectively (0.16 μg/μL); Lane 8 shows 20 μL of protein expressed from the original vector-S.
    Figure Legend Snippet: Detection of purified VP1-hFc protein. Primary antibody used to detect VP1-hFc protein was wt-VP1-immunized rabbit serum (at a dilution of 1:500). HRP-conjugated goat anti-rabbit IgG was diluted to 1:10,000. A: SDS-PAGE analysis of purified VP1-hFc protein. All of the purified VP1-hFc proteins were mixed and concentrated to 1 mL for SDS-PAGE analysis. Different amounts of the protein samples (8 μL, 16 μL and 32 μL; initial concentration of 0.16 μg/μL) were detected. Different amounts of BSA (1 μg, 2 μg and 4 μg) were added as a semi-quantitative gauge. B: ELISA. P1 represents 1 μg of purified and concentrated VP1-hFc protein (0.16 μg/μL) in 100 μL of PBS. Then, P1 was serially diluted 2-fold (P2-P10). Vector-S and VP1-hFc-S represent the original supernatants diluted to 1:5. C: Western blotting assay. Lane 1 shows 20 μL of the original VP1-hFc-S; Lanes 2-7 indicate 40 μL, 20 μL, 10 μL, 5 μL, 2.5 μL and 1.25 μL of purified VP1-hFc protein, respectively (0.16 μg/μL); Lane 8 shows 20 μL of protein expressed from the original vector-S.

    Techniques Used: Purification, SDS Page, Concentration Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot

    Detection of the expression of VP1-hFc protein. The primary antibody used to detect VP1-hFc protein was rabbit anti-human IgG Fc (H+L) used at a dilution of 1:3,000. HRP-conjugated goat anti-rabbit IgG was diluted to 1:10,000. L: lysates, S: supernatants. A: ELISA. Lysates were diluted to 1:10, and supernatants were diluted to 1:5. The results represent the data from 3 biological replicates and are expressed as the mean ± SD. B: Western blotting assay. 20 μL lysates or supernatants was loaded in each lane.
    Figure Legend Snippet: Detection of the expression of VP1-hFc protein. The primary antibody used to detect VP1-hFc protein was rabbit anti-human IgG Fc (H+L) used at a dilution of 1:3,000. HRP-conjugated goat anti-rabbit IgG was diluted to 1:10,000. L: lysates, S: supernatants. A: ELISA. Lysates were diluted to 1:10, and supernatants were diluted to 1:5. The results represent the data from 3 biological replicates and are expressed as the mean ± SD. B: Western blotting assay. 20 μL lysates or supernatants was loaded in each lane.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

    13) Product Images from "Basolateral Targeting of ERBB2 Is Dependent on a Novel Bipartite Juxtamembrane Sorting Signal but Independent of the C-Terminal ERBIN-Binding Domain"

    Article Title: Basolateral Targeting of ERBB2 Is Dependent on a Novel Bipartite Juxtamembrane Sorting Signal but Independent of the C-Terminal ERBIN-Binding Domain

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.18.6553-6563.2002

    ERBB2 contains a juxtamembrane basolateral sorting signal. (A) MDCK cells containing an inducible ERBB2 expression vector were grown on transwell filters to confluence before overnight induction with tetracycline (1 μg/ml). After induction, cells were fixed in methanol, incubated with a rat antibody to ZO-1 and a mouse monoclonal antibody against ERBB2, and subsequently stained with FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rat IgG. Panels show confocal images of the XY sections through the tight junctions, and XZ sections with the apical membrane uppermost. (B) Immunoblot of an MDCK cell clone showing induced and uninduced ERBB2 expression. tet, tetracycline. (C) Schematic depiction of ERBB2 vectors showing the transmembrane domain (TM) and the extent of the C-terminal truncations in the juxtamembrane domain (amino acids 680 to 710). The asterisk shows the position of the introduced termination codon. (D) Immunoblot analysis of truncation mutants analyzed by confocal microscopy in panel E. (E) Confocal microscopy images of XY sections and XZ sections prepared and stained as for panel A. Bars, 5 μm.
    Figure Legend Snippet: ERBB2 contains a juxtamembrane basolateral sorting signal. (A) MDCK cells containing an inducible ERBB2 expression vector were grown on transwell filters to confluence before overnight induction with tetracycline (1 μg/ml). After induction, cells were fixed in methanol, incubated with a rat antibody to ZO-1 and a mouse monoclonal antibody against ERBB2, and subsequently stained with FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rat IgG. Panels show confocal images of the XY sections through the tight junctions, and XZ sections with the apical membrane uppermost. (B) Immunoblot of an MDCK cell clone showing induced and uninduced ERBB2 expression. tet, tetracycline. (C) Schematic depiction of ERBB2 vectors showing the transmembrane domain (TM) and the extent of the C-terminal truncations in the juxtamembrane domain (amino acids 680 to 710). The asterisk shows the position of the introduced termination codon. (D) Immunoblot analysis of truncation mutants analyzed by confocal microscopy in panel E. (E) Confocal microscopy images of XY sections and XZ sections prepared and stained as for panel A. Bars, 5 μm.

    Techniques Used: Expressing, Plasmid Preparation, Incubation, Staining, Confocal Microscopy

    14) Product Images from "Cdc50p, a Conserved Endosomal Membrane Protein, Controls Polarized Growth in Saccharomyces cerevisiae"

    Article Title: Cdc50p, a Conserved Endosomal Membrane Protein, Controls Polarized Growth in Saccharomyces cerevisiae

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-06-0314

    Microscopic observation of GFP-tagged Cdc50p. (A) Punctate localization of Cdc50p-GFP. DIC and GFP images of exponentially growing YKT269 cells containing the CDC50-GFP allele. (B) Colocalization of Cdc50p-GFP with myc-Vps21p. Indirect immunofluorescence was performed as described in MATERIALS AND METHODS. Wild-type cells (YKT38) overexpressing both myc-Vps21p (pSRG92) and Cdc50p-GFP (pKT1266) were grown to midlog phase before fixation. Cdc50p-GFP was visualized by using rabbit polyclonal anti-GFP antibodies and Cy3-conjugated secondary antibodies. The same cells were also stained for myc-Vps21p by using mouse monoclonal anti–c-myc antibody and Cy2-conjugated secondary antibodies. Cy2 and Cy3 images were collected and merged to show coincidence of the two staining patterns. Bar, 5 μm (C) Colocalization of Cdc50p-GFP with FM4–64. Wild-type cells containing the CDC50-GFP allele (YKT271) were grown to midlog phase, labeled in 32 μM FM4–64 for 30 min at 0°C and then chased in fresh medium for 2 min or 12 min at 25°C. Cells were fixed with 1% formaldehyde for 15 min at 25°C. FM4–64 and Cdc50p-GFP images were acquired under the red and green fluorescence channels, respectively. A merged image of these two channels is also shown. Bar, 5 μm. (D) Accumulation of FM4–64 and Cdc50p-GFP in the class E compartment of vps27Δ cells. vps27Δ cells containing the CDC50-GFP allele (YKT569) were grown to midlog phase, labeled in 32 μM FM4–64 for 15 min at 30°C, and then chased in fresh medium for 30 min at 30°C. DIC images of the same cells were collected to visualize the vacuoles. Bar, 5 μm
    Figure Legend Snippet: Microscopic observation of GFP-tagged Cdc50p. (A) Punctate localization of Cdc50p-GFP. DIC and GFP images of exponentially growing YKT269 cells containing the CDC50-GFP allele. (B) Colocalization of Cdc50p-GFP with myc-Vps21p. Indirect immunofluorescence was performed as described in MATERIALS AND METHODS. Wild-type cells (YKT38) overexpressing both myc-Vps21p (pSRG92) and Cdc50p-GFP (pKT1266) were grown to midlog phase before fixation. Cdc50p-GFP was visualized by using rabbit polyclonal anti-GFP antibodies and Cy3-conjugated secondary antibodies. The same cells were also stained for myc-Vps21p by using mouse monoclonal anti–c-myc antibody and Cy2-conjugated secondary antibodies. Cy2 and Cy3 images were collected and merged to show coincidence of the two staining patterns. Bar, 5 μm (C) Colocalization of Cdc50p-GFP with FM4–64. Wild-type cells containing the CDC50-GFP allele (YKT271) were grown to midlog phase, labeled in 32 μM FM4–64 for 30 min at 0°C and then chased in fresh medium for 2 min or 12 min at 25°C. Cells were fixed with 1% formaldehyde for 15 min at 25°C. FM4–64 and Cdc50p-GFP images were acquired under the red and green fluorescence channels, respectively. A merged image of these two channels is also shown. Bar, 5 μm. (D) Accumulation of FM4–64 and Cdc50p-GFP in the class E compartment of vps27Δ cells. vps27Δ cells containing the CDC50-GFP allele (YKT569) were grown to midlog phase, labeled in 32 μM FM4–64 for 15 min at 30°C, and then chased in fresh medium for 30 min at 30°C. DIC images of the same cells were collected to visualize the vacuoles. Bar, 5 μm

    Techniques Used: Immunofluorescence, Staining, Labeling, Fluorescence

    15) Product Images from "Boosting Immunity to Small Tumor-Associated Carbohydrates with Bacteriophage Q? Capsids"

    Article Title: Boosting Immunity to Small Tumor-Associated Carbohydrates with Bacteriophage Q? Capsids

    Journal: ACS chemical biology

    doi: 10.1021/cb400060x

    IgG titer against (a) Qβ virion and (b) BSA-triazole conjugate 14 . Sera are from mice immunized with 1 μg, 4 μg, and 20 μg of Qβ-Tn 7 respectively. The titer numbers were calculated as the highest folds of dilution
    Figure Legend Snippet: IgG titer against (a) Qβ virion and (b) BSA-triazole conjugate 14 . Sera are from mice immunized with 1 μg, 4 μg, and 20 μg of Qβ-Tn 7 respectively. The titer numbers were calculated as the highest folds of dilution

    Techniques Used: Mouse Assay

    Glyco-microarray profiles comparing pre- and post-immune sera from various groups of mice. The average fluorescence signals from at least 4 mice per group are presented (IgM data shown in magenta and IgG data shown in green). Glycans are attached to BSA
    Figure Legend Snippet: Glyco-microarray profiles comparing pre- and post-immune sera from various groups of mice. The average fluorescence signals from at least 4 mice per group are presented (IgM data shown in magenta and IgG data shown in green). Glycans are attached to BSA

    Techniques Used: Microarray, Mouse Assay, Fluorescence

    (A) IgG and (B) IgM responses against Tn antigen in immunized mice with or without exogenous adjuvants. The data shown here are measured at a serum dilution of 1:6400. Each data point represents the optical density at 450 nm measured from serum of individual
    Figure Legend Snippet: (A) IgG and (B) IgM responses against Tn antigen in immunized mice with or without exogenous adjuvants. The data shown here are measured at a serum dilution of 1:6400. Each data point represents the optical density at 450 nm measured from serum of individual

    Techniques Used: Mouse Assay

    (a) IgG and (b) IgM titers against Tn in mice immunized with Qβ-Tn 7, Qβ-propanol 8 and Qβ-glucose 9 . The titer numbers were calculated as the highest folds of dilution that gave three times the absorbance of normal mouse sera
    Figure Legend Snippet: (a) IgG and (b) IgM titers against Tn in mice immunized with Qβ-Tn 7, Qβ-propanol 8 and Qβ-glucose 9 . The titer numbers were calculated as the highest folds of dilution that gave three times the absorbance of normal mouse sera

    Techniques Used: Mouse Assay

    (a) The amounts of Tn and Qβ in six groups of mice for deciphering the importance of antigen display patterns. (b) IgG and (c) IgM responses induced by Qβ conjugated with variable Tn antigen densities (serum dilution 1:6400).
    Figure Legend Snippet: (a) The amounts of Tn and Qβ in six groups of mice for deciphering the importance of antigen display patterns. (b) IgG and (c) IgM responses induced by Qβ conjugated with variable Tn antigen densities (serum dilution 1:6400).

    Techniques Used: Mouse Assay

    16) Product Images from "Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model"

    Article Title: Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.06.030

    (A) High anti-Tn antibody responses are induced by immunization with Qβ-Tn. The primary antibody response is IgG due to significantly higher IgG titers than IgM. The IgG titers from Qβ-Tn are much higher than those from control mice receiving Qβ only. (B) Titers of IgG subtypes antibodies (IgG1, IgG2b, IgG2c, and IgG3) elicited by Qβ-Tn immunization. All major IgG subtypes are induced suggesting balanced IgG responses.
    Figure Legend Snippet: (A) High anti-Tn antibody responses are induced by immunization with Qβ-Tn. The primary antibody response is IgG due to significantly higher IgG titers than IgM. The IgG titers from Qβ-Tn are much higher than those from control mice receiving Qβ only. (B) Titers of IgG subtypes antibodies (IgG1, IgG2b, IgG2c, and IgG3) elicited by Qβ-Tn immunization. All major IgG subtypes are induced suggesting balanced IgG responses.

    Techniques Used: Mouse Assay

    Recognition of (A) TA3Ha cells and (B) Jurkat cells by IgG antibodies in serum from mice before and after immunization with Qβ-Tn (for clarity, only one representative example of binding by postimmune serum is shown). Adapted with permission from Yin, Z., Wright, W. S., McKay, C., Baniel, C., Kaczanowska, K., Bentley, P., et al. (2015). Significant impact of immunogen design on the diversity of antibodies generated by carbohydrate-based anti-cancer vaccine. ACS Chemical Biology , 10 , 2364 – 2372 . Copyright 2015 American Chemical Society.
    Figure Legend Snippet: Recognition of (A) TA3Ha cells and (B) Jurkat cells by IgG antibodies in serum from mice before and after immunization with Qβ-Tn (for clarity, only one representative example of binding by postimmune serum is shown). Adapted with permission from Yin, Z., Wright, W. S., McKay, C., Baniel, C., Kaczanowska, K., Bentley, P., et al. (2015). Significant impact of immunogen design on the diversity of antibodies generated by carbohydrate-based anti-cancer vaccine. ACS Chemical Biology , 10 , 2364 – 2372 . Copyright 2015 American Chemical Society.

    Techniques Used: Mouse Assay, Binding Assay, Generated

    17) Product Images from "A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors"

    Article Title: A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00895

    Comparison of the levels of the apoptotic marker CC3 (A,C) and the proliferation marker Ki67 (B,D) in the tumors of the mice passively immunized with the rabbit IgG against the mPD1-derived mimotope, or in the mice actively immunized with the mimotope, evaluated by IHC staining. For each tumor, more than one region was quantified for detection of evaluated markers. Representative images are shown. Significant differences are indicated by asterisks (* P
    Figure Legend Snippet: Comparison of the levels of the apoptotic marker CC3 (A,C) and the proliferation marker Ki67 (B,D) in the tumors of the mice passively immunized with the rabbit IgG against the mPD1-derived mimotope, or in the mice actively immunized with the mimotope, evaluated by IHC staining. For each tumor, more than one region was quantified for detection of evaluated markers. Representative images are shown. Significant differences are indicated by asterisks (* P

    Techniques Used: Marker, Mouse Assay, Derivative Assay, Immunohistochemistry, Staining

    Evaluation of anti-tumor capacity in vivo by passive immunization with the rabbit IgG against JT–mPD1 in a syngeneic tumor mouse model. (A) Inhibition ELISA showing the binding of mPD–L1 to coated mPD1, before or after preincubation with control rabbit IgG or with different concentrations of rabbit-specific IgG against JT–mPD1 n = 4 for each data point. (B) BALB/c mice either remained untreated (naïve) or were injected as depicted. (C) In vivo anti-tumor effect shown by bars expressing the weight of the tumors explanted upon sacrifice from all the mice in each group. The levels of tumor growth reduction in the mice immunized with the rabbit IgG Abs or the examined mAb against mPD1, compared to the naïve mice, are indicated in percentages above the respective bars. Corresponding macroscopic images of representative explanted tumors are shown below each bar n = 8 for each data point. The results are representative of at least two repeated experiments. Significant differences are indicated by asterisks (** P
    Figure Legend Snippet: Evaluation of anti-tumor capacity in vivo by passive immunization with the rabbit IgG against JT–mPD1 in a syngeneic tumor mouse model. (A) Inhibition ELISA showing the binding of mPD–L1 to coated mPD1, before or after preincubation with control rabbit IgG or with different concentrations of rabbit-specific IgG against JT–mPD1 n = 4 for each data point. (B) BALB/c mice either remained untreated (naïve) or were injected as depicted. (C) In vivo anti-tumor effect shown by bars expressing the weight of the tumors explanted upon sacrifice from all the mice in each group. The levels of tumor growth reduction in the mice immunized with the rabbit IgG Abs or the examined mAb against mPD1, compared to the naïve mice, are indicated in percentages above the respective bars. Corresponding macroscopic images of representative explanted tumors are shown below each bar n = 8 for each data point. The results are representative of at least two repeated experiments. Significant differences are indicated by asterisks (** P

    Techniques Used: In Vivo, Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay, Mouse Assay, Injection, Expressing

    Evaluation of anti-tumor capacity in vivo by active immunization with JT–mPD1 in a syngeneic tumor mouse model. (A) BALB/c mice either remained untreated (naïve) or were immunized as depicted. (B) Level of serum IgG antibody responses against recombinant mPD1 protein, at the time of the sacrifice of the mice immunized with the mimotope. (C) In vivo anti-tumor effect shown by bars expressing the weight of the tumors explanted upon sacrifice from all the mice in each group. The level of tumor growth reduction in the immunized mice, compared to the naïve mice, is indicated in percentages above the respective bar. Corresponding macroscopic images of representative explanted tumors are shown below each bar n = 10 for each data point. The results are representative of at least two repeated experiments. Significant differences are indicated by asterisks (* P
    Figure Legend Snippet: Evaluation of anti-tumor capacity in vivo by active immunization with JT–mPD1 in a syngeneic tumor mouse model. (A) BALB/c mice either remained untreated (naïve) or were immunized as depicted. (B) Level of serum IgG antibody responses against recombinant mPD1 protein, at the time of the sacrifice of the mice immunized with the mimotope. (C) In vivo anti-tumor effect shown by bars expressing the weight of the tumors explanted upon sacrifice from all the mice in each group. The level of tumor growth reduction in the immunized mice, compared to the naïve mice, is indicated in percentages above the respective bar. Corresponding macroscopic images of representative explanted tumors are shown below each bar n = 10 for each data point. The results are representative of at least two repeated experiments. Significant differences are indicated by asterisks (* P

    Techniques Used: In Vivo, Mouse Assay, Recombinant, Expressing

    18) Product Images from "Cowpea Mosaic Virus Capsid, a Promising Carrier for the Development of Carbohydrate Based Anti-tumor Vaccines"

    Article Title: Cowpea Mosaic Virus Capsid, a Promising Carrier for the Development of Carbohydrate Based Anti-tumor Vaccines

    Journal: Chemistry (Weinheim an der Bergstrasse, Germany)

    doi: 10.1002/chem.200

    a) ELISA titers of anti-Tn IgG+IgM antibody in mouse sera prior to immunization (day 0), after immunization with Tn-1-S-CPMV (day 35) in the absence and presence of 0.1 M GalNAc. b) ELISA titers of anti-Tn IgM and IgG antibody in mouse sera after immunization
    Figure Legend Snippet: a) ELISA titers of anti-Tn IgG+IgM antibody in mouse sera prior to immunization (day 0), after immunization with Tn-1-S-CPMV (day 35) in the absence and presence of 0.1 M GalNAc. b) ELISA titers of anti-Tn IgM and IgG antibody in mouse sera after immunization

    Techniques Used: Enzyme-linked Immunosorbent Assay

    19) Product Images from "Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus"

    Article Title: Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0227109

    Immunization of mice with the live-attenuated double mutant SCV (Δ vraG Δ hemB ) stimulates a strong humoral response against S . aureus . The six groups of mice (n = 5 mice per group) are defined in the Materials and Methods section and the immunization schedule is shown in Fig 1 . Sera were collected before priming immunization (preimmune) and 10 days after the boost immunization (immune). Each dot represents the total IgG titer of one mouse against S . aureus Δ vraG Δ hemB whole cell extract, and boxes (upper and lower boundaries) and vertical lines (whiskers) indicate interquartile distances and ranges, respectively. Medians are represented by thick horizontal lines for immune titers and dashed lines for preimmune titers. Immune titers were compared to their corresponding preimmune titers (Two-way ANOVA and Sidak's multiple comparisons test: φφφφ P ≤ 0.0001; φφφ P ≤ 0.001; NS, not significant), as indicated on the top of the corresponding box, or to other groups (Two-way ANOVA and Tukey's multiple comparisons test: ****P ≤ 0.0001; NS, not significant).
    Figure Legend Snippet: Immunization of mice with the live-attenuated double mutant SCV (Δ vraG Δ hemB ) stimulates a strong humoral response against S . aureus . The six groups of mice (n = 5 mice per group) are defined in the Materials and Methods section and the immunization schedule is shown in Fig 1 . Sera were collected before priming immunization (preimmune) and 10 days after the boost immunization (immune). Each dot represents the total IgG titer of one mouse against S . aureus Δ vraG Δ hemB whole cell extract, and boxes (upper and lower boundaries) and vertical lines (whiskers) indicate interquartile distances and ranges, respectively. Medians are represented by thick horizontal lines for immune titers and dashed lines for preimmune titers. Immune titers were compared to their corresponding preimmune titers (Two-way ANOVA and Sidak's multiple comparisons test: φφφφ P ≤ 0.0001; φφφ P ≤ 0.001; NS, not significant), as indicated on the top of the corresponding box, or to other groups (Two-way ANOVA and Tukey's multiple comparisons test: ****P ≤ 0.0001; NS, not significant).

    Techniques Used: Mouse Assay, Mutagenesis

    Th1/Th2 immune response balance of mice immunized with the live-attenuated double mutant SCV (Δ vraG Δ hemB ) or inactivated bacteria. The six groups of mice shown in (A), (B) and (C) are defined in the Materials and Methods section and the immunization schedule is shown in Fig 1 . Specific IgG2a/IgG1 ratios (A) and IgG2a and IgG1 titers (B-C) of mice against Δ vraG Δ hemB whole cell extracts. Each dot represents the IgG2a/Ig1 ratio or the immune IgG2a and IgG1 titer of one mouse. Ratios were calculated using the specific IgG2a/IgG1 titers of each mouse. Medians are represented by horizontal lines. Ratios or titers were compared between each group (A: Kruskal-Wallis test with Dunn’s multiple comparison test; *P ≤ 0.05; B-C: Two-way ANOVA and Sidak's multiple comparisons test; ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01; NS, not significant).
    Figure Legend Snippet: Th1/Th2 immune response balance of mice immunized with the live-attenuated double mutant SCV (Δ vraG Δ hemB ) or inactivated bacteria. The six groups of mice shown in (A), (B) and (C) are defined in the Materials and Methods section and the immunization schedule is shown in Fig 1 . Specific IgG2a/IgG1 ratios (A) and IgG2a and IgG1 titers (B-C) of mice against Δ vraG Δ hemB whole cell extracts. Each dot represents the IgG2a/Ig1 ratio or the immune IgG2a and IgG1 titer of one mouse. Ratios were calculated using the specific IgG2a/IgG1 titers of each mouse. Medians are represented by horizontal lines. Ratios or titers were compared between each group (A: Kruskal-Wallis test with Dunn’s multiple comparison test; *P ≤ 0.05; B-C: Two-way ANOVA and Sidak's multiple comparisons test; ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01; NS, not significant).

    Techniques Used: Mouse Assay, Mutagenesis

    20) Product Images from "Tobacco Mosaic Virus as a New Carrier for Tumor Associated Carbohydrate Antigens"

    Article Title: Tobacco Mosaic Virus as a New Carrier for Tumor Associated Carbohydrate Antigens

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc300244a

    a) ELISA titers of anti-Tn IgG + IgM antibodies for control group TMV, and TMV-Tyr-Tn (20 μg Tn) with either CFA or TiterMax Gold adjuvant; b) ELISA titers of anti-Tn IgG + IgM antibodies generated by TMV-Tyr-Tn with either CFA or TiterMax Gold in the absence and presence of 0.1 M GalNAc; c) ELISA titers of anti-Tn IgG and IgM antibodies.
    Figure Legend Snippet: a) ELISA titers of anti-Tn IgG + IgM antibodies for control group TMV, and TMV-Tyr-Tn (20 μg Tn) with either CFA or TiterMax Gold adjuvant; b) ELISA titers of anti-Tn IgG + IgM antibodies generated by TMV-Tyr-Tn with either CFA or TiterMax Gold in the absence and presence of 0.1 M GalNAc; c) ELISA titers of anti-Tn IgG and IgM antibodies.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Generated

    Anti-Tn ELISA titers of a) IgG + IgM for TMV control group and TMV-OEG-Tn groups corresponding to 1 μg, 4 μg, and 20 μg Tn respectively; b) IgG and c) IgM of TMV-OEG-Tn groups corresponding to 1 μg, 4 μg, and 20 μg Tn respectively.
    Figure Legend Snippet: Anti-Tn ELISA titers of a) IgG + IgM for TMV control group and TMV-OEG-Tn groups corresponding to 1 μg, 4 μg, and 20 μg Tn respectively; b) IgG and c) IgM of TMV-OEG-Tn groups corresponding to 1 μg, 4 μg, and 20 μg Tn respectively.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    21) Product Images from "Calreticulin Couples Calcium Release and Calcium Influx in Integrin-mediated Calcium Signaling"

    Article Title: Calreticulin Couples Calcium Release and Calcium Influx in Integrin-mediated Calcium Signaling

    Journal: Molecular Biology of the Cell

    doi:

    Association of integrin α7 with ectocalreticulin and DHPR α1 subunit in E63 cells. (A) The unfixed E63 cells (5 d old) were first immunostained with calreticulin Ab (PA3-900) and then stained with integrin α7 Ab (O26 mAb) again. Ectocalreticulin was diffusely distributed on the cell surface (a), whereas integrin α7 displayed a stress fiber-like distribution (b). In contrast, prior incubation of integrin α7 Ab (c–h) before staining of calreticulin Ab (PA3-900 [c and g] or LAR090 [e]) induced cellular localization of ectocalreticulin (c, e, and g), in which ectocalreticulin appears to be colocalized with integrin α7 (d, f, and h). In control experiments, cells were first stained with normal mouse immunoglobulin and then stained with normal rabbit immunoglobulin (i and j). Cells shown in the left panels (a, c, e, g, and i) were stained with TRITC-conjugated donkey anti-rabbit immunoglobulin, whereas those in the right panels (b, d, f, h, and j) were stained with FITC-conjugated donkey anti-mouse immunoglobulin. Bars: in f (for a–f, i, and j), 10 μm; in h (for g and j), 5 μm. (B) Cell surface proteins in 5-d cultured cells were iodinated with Na 125 I and immunoprecipitated with calreticulin Ab (PA3-900). Autoradiography of cell surface proteins (a) and imunoblot analysis (b) of cell lysates with calreticulin Ab (PA3-900) are presented. Iodinated ectocalreticulin is indicated by 125 I-CRT. (C) Cell lysates were immunoprecipitated (IP) using integrin α7 Ab (H36 mAb) and DHPR α1 Ab and then immunoblotted (IB) with calreticulin Ab: PA3-900 (left panel) and LAR090 (right panel). (D) After stripping, the same membrane was reprobed with DHPR α1 Ab. Note that integrin α7 was associated with both calreticulin and DHPR α1.
    Figure Legend Snippet: Association of integrin α7 with ectocalreticulin and DHPR α1 subunit in E63 cells. (A) The unfixed E63 cells (5 d old) were first immunostained with calreticulin Ab (PA3-900) and then stained with integrin α7 Ab (O26 mAb) again. Ectocalreticulin was diffusely distributed on the cell surface (a), whereas integrin α7 displayed a stress fiber-like distribution (b). In contrast, prior incubation of integrin α7 Ab (c–h) before staining of calreticulin Ab (PA3-900 [c and g] or LAR090 [e]) induced cellular localization of ectocalreticulin (c, e, and g), in which ectocalreticulin appears to be colocalized with integrin α7 (d, f, and h). In control experiments, cells were first stained with normal mouse immunoglobulin and then stained with normal rabbit immunoglobulin (i and j). Cells shown in the left panels (a, c, e, g, and i) were stained with TRITC-conjugated donkey anti-rabbit immunoglobulin, whereas those in the right panels (b, d, f, h, and j) were stained with FITC-conjugated donkey anti-mouse immunoglobulin. Bars: in f (for a–f, i, and j), 10 μm; in h (for g and j), 5 μm. (B) Cell surface proteins in 5-d cultured cells were iodinated with Na 125 I and immunoprecipitated with calreticulin Ab (PA3-900). Autoradiography of cell surface proteins (a) and imunoblot analysis (b) of cell lysates with calreticulin Ab (PA3-900) are presented. Iodinated ectocalreticulin is indicated by 125 I-CRT. (C) Cell lysates were immunoprecipitated (IP) using integrin α7 Ab (H36 mAb) and DHPR α1 Ab and then immunoblotted (IB) with calreticulin Ab: PA3-900 (left panel) and LAR090 (right panel). (D) After stripping, the same membrane was reprobed with DHPR α1 Ab. Note that integrin α7 was associated with both calreticulin and DHPR α1.

    Techniques Used: Staining, Incubation, Cell Culture, Immunoprecipitation, Autoradiography, Stripping Membranes

    22) Product Images from "Balanced splicing at the Tat-specific HIV-1 3′ss A3 is critical for HIV-1 replication"

    Article Title: Balanced splicing at the Tat-specific HIV-1 3′ss A3 is critical for HIV-1 replication

    Journal: Retrovirology

    doi: 10.1186/s12977-015-0154-8

    The antiviral activity of the SRSF6 variants correlates with their subcellular localization. Subcellular localization of the HA-tagged SRSF6 variants was unraveled by immunostaining of transiently transfected HeLa cells using an anti-HA and an anti-mouse Alexa Fluor® 488 antibody. Cell nuclei were stained with DAPI and glass slides were analyzed by confocal laser-scanning microscopy.
    Figure Legend Snippet: The antiviral activity of the SRSF6 variants correlates with their subcellular localization. Subcellular localization of the HA-tagged SRSF6 variants was unraveled by immunostaining of transiently transfected HeLa cells using an anti-HA and an anti-mouse Alexa Fluor® 488 antibody. Cell nuclei were stained with DAPI and glass slides were analyzed by confocal laser-scanning microscopy.

    Techniques Used: Activity Assay, Immunostaining, Transfection, Staining, Confocal Laser Scanning Microscopy

    Model for SRSF2 and SRSF6-mediated 3′ss activation. (A) SRSF6 and hnRNP A/B proteins compete for binding to their overlapping binding sites located within ESE2 and ESS2. Depending on the SRSF6 binding efficiency (B) , SRSF2 interacts with the upstream ESE tat and guides spliceosomal components to 3′ss A3. (C) Inactivation of ESE tat is associated with a failure in efficient spliceosome recruitment irrespective of higher SRSF6 binding (D) .
    Figure Legend Snippet: Model for SRSF2 and SRSF6-mediated 3′ss activation. (A) SRSF6 and hnRNP A/B proteins compete for binding to their overlapping binding sites located within ESE2 and ESS2. Depending on the SRSF6 binding efficiency (B) , SRSF2 interacts with the upstream ESE tat and guides spliceosomal components to 3′ss A3. (C) Inactivation of ESE tat is associated with a failure in efficient spliceosome recruitment irrespective of higher SRSF6 binding (D) .

    Techniques Used: Activation Assay, Binding Assay

    The SR proteins SRSF2 and SRSF6 bind downstream of 3′ss A3 to control tat -mRNA splicing. (A) In vitro -transcribed RNA substrates used for the RNA pull-down assays. Mutated nucleotides are indicated below the wild-type reference (pNL4-3) at corresponding positions and “-“ denotes wild type nucleotide. (B) RNAs were immobilized on Agarose beads and analyzed for the presence of SR proteins with specific antibodies directed against SRSF2 (Abcam, ab28428) or phosphorylated SR proteins (Invitrogen, 1H4G7). Recombinant MS2 coat protein was added to HeLa cell nuclear extracts and served as a control for equal precipitation efficiencies. (C) 2.5 × 10 5 HEK293T were transfected with pNL4-3 or mutant provirus and pcDNA3.1(+), an SRSF2 or SRSF6-expressing plasmid. 48 h after transfection RNAs were analyzed by RT-PCR (C) or Northern blot (D) . (E) Cell lysates and supernatants from transfected HEK293T cells were analyzed for Gag expression as described before (see Figure 2 ). (F) Left: Cell lysates and supernatants from HEK293T cells cotransfected with pNL4-3 and gradually increasing amounts of SRSF6 expressing plasmid that were analyzed for Gag expression as described before. Right: Serial dilutions of cell lysates and supernatants from transfected HEK293T cells analyzed for Gag expression.
    Figure Legend Snippet: The SR proteins SRSF2 and SRSF6 bind downstream of 3′ss A3 to control tat -mRNA splicing. (A) In vitro -transcribed RNA substrates used for the RNA pull-down assays. Mutated nucleotides are indicated below the wild-type reference (pNL4-3) at corresponding positions and “-“ denotes wild type nucleotide. (B) RNAs were immobilized on Agarose beads and analyzed for the presence of SR proteins with specific antibodies directed against SRSF2 (Abcam, ab28428) or phosphorylated SR proteins (Invitrogen, 1H4G7). Recombinant MS2 coat protein was added to HeLa cell nuclear extracts and served as a control for equal precipitation efficiencies. (C) 2.5 × 10 5 HEK293T were transfected with pNL4-3 or mutant provirus and pcDNA3.1(+), an SRSF2 or SRSF6-expressing plasmid. 48 h after transfection RNAs were analyzed by RT-PCR (C) or Northern blot (D) . (E) Cell lysates and supernatants from transfected HEK293T cells were analyzed for Gag expression as described before (see Figure 2 ). (F) Left: Cell lysates and supernatants from HEK293T cells cotransfected with pNL4-3 and gradually increasing amounts of SRSF6 expressing plasmid that were analyzed for Gag expression as described before. Right: Serial dilutions of cell lysates and supernatants from transfected HEK293T cells analyzed for Gag expression.

    Techniques Used: In Vitro, Recombinant, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Northern Blot

    The arginine-serine (RS) rich C-terminus as well as the RRMH domain of SRSF6 are dispensable for its antiviral activity. (A) SRSF6 protein variants used in the coexpression experiments. Proper expression of all truncated SRSF6 variants was confirmed using an antibody specifically recognizing an HA epitope (Sigma-Aldrich, H6908), which was C-terminally fused to each mutant. HEK293T cells were transiently cotransfected with 1 μg of pNL4-3 and 1 μg of pcDNA3.1(+) or the respective SRSF6 variant-expression plasmid. Samples from the same RNA preparations were analyzed by both RT-PCR (B) and Northern blot (C) . (D) Cellular lysates and supernatants were analyzed by Western blot using antibodies directed against viral Gag or cellular actin (loading control). Values and error bars show the average ± standard deviation of two independent transfection experiments.
    Figure Legend Snippet: The arginine-serine (RS) rich C-terminus as well as the RRMH domain of SRSF6 are dispensable for its antiviral activity. (A) SRSF6 protein variants used in the coexpression experiments. Proper expression of all truncated SRSF6 variants was confirmed using an antibody specifically recognizing an HA epitope (Sigma-Aldrich, H6908), which was C-terminally fused to each mutant. HEK293T cells were transiently cotransfected with 1 μg of pNL4-3 and 1 μg of pcDNA3.1(+) or the respective SRSF6 variant-expression plasmid. Samples from the same RNA preparations were analyzed by both RT-PCR (B) and Northern blot (C) . (D) Cellular lysates and supernatants were analyzed by Western blot using antibodies directed against viral Gag or cellular actin (loading control). Values and error bars show the average ± standard deviation of two independent transfection experiments.

    Techniques Used: Activity Assay, Expressing, Mutagenesis, Variant Assay, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Western Blot, Standard Deviation, Transfection

    23) Product Images from "Complete Removal of Extracellular IgG Antibodies in a Randomized Dose-Escalation Phase I Study with the Bacterial Enzyme IdeS – A Novel Therapeutic Opportunity"

    Article Title: Complete Removal of Extracellular IgG Antibodies in a Randomized Dose-Escalation Phase I Study with the Bacterial Enzyme IdeS – A Novel Therapeutic Opportunity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0132011

    Quantitative pharmacodynamics analysis by ELISA showed rapid degradation of IgG. Serum IgG levels from all four individual subjects dosed with 0.12 mg/kg BW IdeS (A and B) and all four individual subjects dosed with 0.24 mg/kg BW IdeS (C and D) determined using a validated ELISA method performed by Covance Laboratories Ltd, UK. To be able to follow both early, rapid degradation as well as recovery of IgG, graphs A and C show data up to 48 hours after dosing (x-axis in hours) and graphs B and D show data until last visit (x-axis in days).
    Figure Legend Snippet: Quantitative pharmacodynamics analysis by ELISA showed rapid degradation of IgG. Serum IgG levels from all four individual subjects dosed with 0.12 mg/kg BW IdeS (A and B) and all four individual subjects dosed with 0.24 mg/kg BW IdeS (C and D) determined using a validated ELISA method performed by Covance Laboratories Ltd, UK. To be able to follow both early, rapid degradation as well as recovery of IgG, graphs A and C show data up to 48 hours after dosing (x-axis in hours) and graphs B and D show data until last visit (x-axis in days).

    Techniques Used: Enzyme-linked Immunosorbent Assay

    24) Product Images from "Incorporating B cell activating factor (BAFF) into the membrane of rabies virus (RABV) particles improves the speed and magnitude of vaccine-induced antibody responses"

    Article Title: Incorporating B cell activating factor (BAFF) into the membrane of rabies virus (RABV) particles improves the speed and magnitude of vaccine-induced antibody responses

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007800

    100-fold less RABV-ED51-mBAFF is needed to induce comparable immunity in mice immunized with RABV. C57BL/6J mice were immunized i.m. with 10 3 or 10 5 ffu/mouse of RABV or RABV-ED51-mBAFF, or PBS as a negative control. Blood was collected on days 5, 7 and 10 post-immunization as a source of serum. Four three-fold serial dilutions of sera were analyzed by ELISA to determine anti-RABV G IgM (A and B) or IgG (C and D) antibody titers and presented as OD 490 of the reciprocal serial dilution. For comparison, sera from PBS-immunized mice were tested in parallel. Statistical difference in antibody titers by ELISA between RABV- and RABV-ED51-mBAFF-immunized mice was determined using an unpaired, two-tailed t test and data is presented as the mean ± SEM. *p≤0.05; **p ≤ 0.01; ***p≤0.001; ****p≤0.0001; N = 10 mice/group). (ffu = focus forming units; OD = optical density).
    Figure Legend Snippet: 100-fold less RABV-ED51-mBAFF is needed to induce comparable immunity in mice immunized with RABV. C57BL/6J mice were immunized i.m. with 10 3 or 10 5 ffu/mouse of RABV or RABV-ED51-mBAFF, or PBS as a negative control. Blood was collected on days 5, 7 and 10 post-immunization as a source of serum. Four three-fold serial dilutions of sera were analyzed by ELISA to determine anti-RABV G IgM (A and B) or IgG (C and D) antibody titers and presented as OD 490 of the reciprocal serial dilution. For comparison, sera from PBS-immunized mice were tested in parallel. Statistical difference in antibody titers by ELISA between RABV- and RABV-ED51-mBAFF-immunized mice was determined using an unpaired, two-tailed t test and data is presented as the mean ± SEM. *p≤0.05; **p ≤ 0.01; ***p≤0.001; ****p≤0.0001; N = 10 mice/group). (ffu = focus forming units; OD = optical density).

    Techniques Used: Mouse Assay, Negative Control, Enzyme-linked Immunosorbent Assay, Serial Dilution, Two Tailed Test

    25) Product Images from "Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever"

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06741-w

    Analysis of the humoral response towards Lassa virus glycoprotein. C57BL/6 mice were immunized IM in the gastrocnemius muscle with either 10 μg of β-propiolactone inactivated viral particles in PBS or adjuvanted with 5 μg of GLA, a TLR-4 agonist formulated in 2% of stable emulsion (SE); LASSARAB+GLA-SE, LASSARAB, FILORAB1 groups) and boosted two times with the same amount on day 7 and 28 ( a ). Immunizations with replication-competent viruses were executed with a single time inoculation of 10 6 ffu or pfu virus IM in the gastrocnemius (rc-LASSARAB; rc-FILORAB1 groups and rVSV-GPC). b The EC 50 values (obtained from the 4PL regression ELISA curve) of the total IgG titers against LASV GPC are plotted since day 0 until day 42. Error bars are representative of the standard error mean (SEM) and is calculated from 15 mice per group. Statistical significance was calculated by using 2-way ANOVA–post-hoc Tukey’s Honest Significant Difference Test. c ELISA of total IgG against LASV GPC of all day 42 groups are shown for all immunized groups. ELISA curves are generated from 4PL regression. Error bars are representative of the SEM of OD 490 values (five mice per group, in triplicates). d Day 35 EC 50 antibody titer of IgG sub-isotype (IgG2c and IgG1) against LASV GPC of sera from LASSARAB+GLA-SE and LASSARAB group was analyzed. Error bars are the SEM of a total of five mice per group and statistical significance by 2-way ANOVA (post-hoc Tukey’s Honest Significant Difference Test). e The ratios of the respective EC 50 antibody titers IgG1/IgG2c are plotted and the F test was applied to check for variance difference ( p
    Figure Legend Snippet: Analysis of the humoral response towards Lassa virus glycoprotein. C57BL/6 mice were immunized IM in the gastrocnemius muscle with either 10 μg of β-propiolactone inactivated viral particles in PBS or adjuvanted with 5 μg of GLA, a TLR-4 agonist formulated in 2% of stable emulsion (SE); LASSARAB+GLA-SE, LASSARAB, FILORAB1 groups) and boosted two times with the same amount on day 7 and 28 ( a ). Immunizations with replication-competent viruses were executed with a single time inoculation of 10 6 ffu or pfu virus IM in the gastrocnemius (rc-LASSARAB; rc-FILORAB1 groups and rVSV-GPC). b The EC 50 values (obtained from the 4PL regression ELISA curve) of the total IgG titers against LASV GPC are plotted since day 0 until day 42. Error bars are representative of the standard error mean (SEM) and is calculated from 15 mice per group. Statistical significance was calculated by using 2-way ANOVA–post-hoc Tukey’s Honest Significant Difference Test. c ELISA of total IgG against LASV GPC of all day 42 groups are shown for all immunized groups. ELISA curves are generated from 4PL regression. Error bars are representative of the SEM of OD 490 values (five mice per group, in triplicates). d Day 35 EC 50 antibody titer of IgG sub-isotype (IgG2c and IgG1) against LASV GPC of sera from LASSARAB+GLA-SE and LASSARAB group was analyzed. Error bars are the SEM of a total of five mice per group and statistical significance by 2-way ANOVA (post-hoc Tukey’s Honest Significant Difference Test). e The ratios of the respective EC 50 antibody titers IgG1/IgG2c are plotted and the F test was applied to check for variance difference ( p

    Techniques Used: Mouse Assay, Gel Permeation Chromatography, Enzyme-linked Immunosorbent Assay, Generated

    26) Product Images from "Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever"

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06741-w

    Analysis of the humoral response towards Lassa virus glycoprotein. C57BL/6 mice were immunized IM in the gastrocnemius muscle with either 10 μg of β-propiolactone inactivated viral particles in PBS or adjuvanted with 5 μg of GLA, a TLR-4 agonist formulated in 2% of stable emulsion (SE); LASSARAB+GLA-SE, LASSARAB, FILORAB1 groups) and boosted two times with the same amount on day 7 and 28 ( a ). Immunizations with replication-competent viruses were executed with a single time inoculation of 10 6 ffu or pfu virus IM in the gastrocnemius (rc-LASSARAB; rc-FILORAB1 groups and rVSV-GPC). b The EC 50 values (obtained from the 4PL regression ELISA curve) of the total IgG titers against LASV GPC are plotted since day 0 until day 42. Error bars are representative of the standard error mean (SEM) and is calculated from 15 mice per group. Statistical significance was calculated by using 2-way ANOVA–post-hoc Tukey’s Honest Significant Difference Test. c ELISA of total IgG against LASV GPC of all day 42 groups are shown for all immunized groups. ELISA curves are generated from 4PL regression. Error bars are representative of the SEM of OD 490 values (five mice per group, in triplicates). d Day 35 EC 50 antibody titer of IgG sub-isotype (IgG2c and IgG1) against LASV GPC of sera from LASSARAB+GLA-SE and LASSARAB group was analyzed. Error bars are the SEM of a total of five mice per group and statistical significance by 2-way ANOVA (post-hoc Tukey’s Honest Significant Difference Test). e The ratios of the respective EC 50 antibody titers IgG1/IgG2c are plotted and the F test was applied to check for variance difference ( p
    Figure Legend Snippet: Analysis of the humoral response towards Lassa virus glycoprotein. C57BL/6 mice were immunized IM in the gastrocnemius muscle with either 10 μg of β-propiolactone inactivated viral particles in PBS or adjuvanted with 5 μg of GLA, a TLR-4 agonist formulated in 2% of stable emulsion (SE); LASSARAB+GLA-SE, LASSARAB, FILORAB1 groups) and boosted two times with the same amount on day 7 and 28 ( a ). Immunizations with replication-competent viruses were executed with a single time inoculation of 10 6 ffu or pfu virus IM in the gastrocnemius (rc-LASSARAB; rc-FILORAB1 groups and rVSV-GPC). b The EC 50 values (obtained from the 4PL regression ELISA curve) of the total IgG titers against LASV GPC are plotted since day 0 until day 42. Error bars are representative of the standard error mean (SEM) and is calculated from 15 mice per group. Statistical significance was calculated by using 2-way ANOVA–post-hoc Tukey’s Honest Significant Difference Test. c ELISA of total IgG against LASV GPC of all day 42 groups are shown for all immunized groups. ELISA curves are generated from 4PL regression. Error bars are representative of the SEM of OD 490 values (five mice per group, in triplicates). d Day 35 EC 50 antibody titer of IgG sub-isotype (IgG2c and IgG1) against LASV GPC of sera from LASSARAB+GLA-SE and LASSARAB group was analyzed. Error bars are the SEM of a total of five mice per group and statistical significance by 2-way ANOVA (post-hoc Tukey’s Honest Significant Difference Test). e The ratios of the respective EC 50 antibody titers IgG1/IgG2c are plotted and the F test was applied to check for variance difference ( p

    Techniques Used: Mouse Assay, Gel Permeation Chromatography, Enzyme-linked Immunosorbent Assay, Generated

    27) Product Images from "Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections"

    Article Title: Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0166621

    Immunization of mice with the live-attenuated double mutant (Δ720Δ hemB ) induces a strong humoral response against S . aureus bovine mastitis isolates and against a specific cell-wall associated antigen (IsdH). Mice were immunized as previously described: serums were collected before priming immunization (preimmune, open circles) and ten days after the boost immunization (immune, blue squares). A. IgG titers rise with increasing immunization doses (10 6 , 10 7 , 10 8 CFU) of the live-attenuated mutant Δ vraG Δ hemB : each dot represents the total IgG titer of one mouse against a Δ vraG Δ hemB whole cell extract. Medians are represented by thick lines for immune titers and dashed lines for preimmune titers. Titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey’s multiple comparisons test: ****: P ≤ 0.0001). B. Immunization with the live-attenuated mutant Δ vraG Δ hemB confers high IgG titers against components that are shared by mastitis strains of commonly found spa types. Each dot represents the total IgG titer of one mouse against the whole cell extract of the indicated strain. Medians are represented by thick lines for immune titers and dashed lines for preimmune titers. All immune titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey’s multiple comparisons test: P ≤ 0.0001 for all groups). C. Immunization with the live-attenuated mutant Δ vraG Δ hemB confers specific IgG titers against the cell-wall associated protein IsdH. Each dot represents the total IgG titer of one mouse against recombinant IsdH. Compared groups were immunized with the 10 8 CFU of the live-attenuated Δ vraG Δ hemB (ΔΔ) or 25 μg of the purified recombinant IsdH protein (IsdH). D. IgG isotype ratios (IgG2a/IgG1) of mice immunized with the live-attenuated mutant Δ vraG Δ hemB (open diamonds) or immunized with the recombinant IsdH (black diamonds), against whole-cell extracts of strain Δ vraG Δ hemB (vs ΔΔ) or against the recombinant IsdH protein (vs IsdH). Each diamond represents the IgG2a/IgG1 titer ratio for one mouse. Medians are represented by thick lines (One-way ANOVA and Dunn’s multiple comparison test: *: P ≤ 0.05).
    Figure Legend Snippet: Immunization of mice with the live-attenuated double mutant (Δ720Δ hemB ) induces a strong humoral response against S . aureus bovine mastitis isolates and against a specific cell-wall associated antigen (IsdH). Mice were immunized as previously described: serums were collected before priming immunization (preimmune, open circles) and ten days after the boost immunization (immune, blue squares). A. IgG titers rise with increasing immunization doses (10 6 , 10 7 , 10 8 CFU) of the live-attenuated mutant Δ vraG Δ hemB : each dot represents the total IgG titer of one mouse against a Δ vraG Δ hemB whole cell extract. Medians are represented by thick lines for immune titers and dashed lines for preimmune titers. Titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey’s multiple comparisons test: ****: P ≤ 0.0001). B. Immunization with the live-attenuated mutant Δ vraG Δ hemB confers high IgG titers against components that are shared by mastitis strains of commonly found spa types. Each dot represents the total IgG titer of one mouse against the whole cell extract of the indicated strain. Medians are represented by thick lines for immune titers and dashed lines for preimmune titers. All immune titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey’s multiple comparisons test: P ≤ 0.0001 for all groups). C. Immunization with the live-attenuated mutant Δ vraG Δ hemB confers specific IgG titers against the cell-wall associated protein IsdH. Each dot represents the total IgG titer of one mouse against recombinant IsdH. Compared groups were immunized with the 10 8 CFU of the live-attenuated Δ vraG Δ hemB (ΔΔ) or 25 μg of the purified recombinant IsdH protein (IsdH). D. IgG isotype ratios (IgG2a/IgG1) of mice immunized with the live-attenuated mutant Δ vraG Δ hemB (open diamonds) or immunized with the recombinant IsdH (black diamonds), against whole-cell extracts of strain Δ vraG Δ hemB (vs ΔΔ) or against the recombinant IsdH protein (vs IsdH). Each diamond represents the IgG2a/IgG1 titer ratio for one mouse. Medians are represented by thick lines (One-way ANOVA and Dunn’s multiple comparison test: *: P ≤ 0.05).

    Techniques Used: Mouse Assay, Mutagenesis, Recombinant, Purification

    28) Product Images from "Nidogen-1 is a novel extracellular ligand for the NKp44 activating receptor"

    Article Title: Nidogen-1 is a novel extracellular ligand for the NKp44 activating receptor

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1470730

    Specific recognition of NID1 by NKp44Fc chimeric receptor. (A) Concentrated HEK293T-SN was immunoprecipitated with the indicated Fc molecules and analyzed in 7.5% SDS-PAGE under non-reducing conditions; in parallel, concentrated HEK293T-SN was loaded as a positive control. After blotting, the membrane was probed with mouse anti-NID1 mAb followed by HRP-conjugated anti-mouse IgG mAb. One representative experiment of three is shown. (B) Concentrated HEK293T-SN and rNID1 were analyzed in SDS-PAGE on a 7.5% polyacrylamide gel under non-reducing conditions and, after blotting, probed with the indicated Fc molecules or with anti-NID1 mAb, followed by HRP-conjugated anti-human or anti-mouse IgG secondary reagent, respectively. Molecular weight (MW) markers (kDa) are indicated on the right. One representative experiment of five is shown. (C) ELISA plates were coated with rNID1 and incubated with different concentrations of the indicated Fc molecules followed by HRP-conjugated anti-human IgG mAb. Graph represents absorbance at 405 nm after normalization to background (nonspecific binding of the secondary reagent). Data are medians of triplicates ± interquartile range and are the pooled results of three independent experiments. **** p
    Figure Legend Snippet: Specific recognition of NID1 by NKp44Fc chimeric receptor. (A) Concentrated HEK293T-SN was immunoprecipitated with the indicated Fc molecules and analyzed in 7.5% SDS-PAGE under non-reducing conditions; in parallel, concentrated HEK293T-SN was loaded as a positive control. After blotting, the membrane was probed with mouse anti-NID1 mAb followed by HRP-conjugated anti-mouse IgG mAb. One representative experiment of three is shown. (B) Concentrated HEK293T-SN and rNID1 were analyzed in SDS-PAGE on a 7.5% polyacrylamide gel under non-reducing conditions and, after blotting, probed with the indicated Fc molecules or with anti-NID1 mAb, followed by HRP-conjugated anti-human or anti-mouse IgG secondary reagent, respectively. Molecular weight (MW) markers (kDa) are indicated on the right. One representative experiment of five is shown. (C) ELISA plates were coated with rNID1 and incubated with different concentrations of the indicated Fc molecules followed by HRP-conjugated anti-human IgG mAb. Graph represents absorbance at 405 nm after normalization to background (nonspecific binding of the secondary reagent). Data are medians of triplicates ± interquartile range and are the pooled results of three independent experiments. **** p

    Techniques Used: Immunoprecipitation, SDS Page, Positive Control, Molecular Weight, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

    NKp44Fc recognizes NID1 released by NID1-transfected K562 cells. (A) NID1 mRNA expression was assessed by RT-PCR in wild type or NID1-transfected K562 cells. Primers specific for β-actin were utilized as positive control. PCR products were run on 1.5% agarose gel and visualized by ethidium bromide staining. One representative experiment of three is shown. (B, C) ELISA plates were coated with mouse anti-NID1 mAb (B) or with the indicated Fc molecules (C), followed by incubation with concentrated SN obtained from untransfected or NID1-transfected K562 cells cultured in protein-free medium. NID1 was detected using a goat anti-NID1 Ab followed by a HRP-conjugated anti-goat IgG Ab. Graphs represent absorbance at 405 nm after normalization to background (nonspecific binding of goat anti-NID1 + secondary reagent). Data are medians of triplicates ± interquartile range and are the pooled results of three independent experiments. **** p
    Figure Legend Snippet: NKp44Fc recognizes NID1 released by NID1-transfected K562 cells. (A) NID1 mRNA expression was assessed by RT-PCR in wild type or NID1-transfected K562 cells. Primers specific for β-actin were utilized as positive control. PCR products were run on 1.5% agarose gel and visualized by ethidium bromide staining. One representative experiment of three is shown. (B, C) ELISA plates were coated with mouse anti-NID1 mAb (B) or with the indicated Fc molecules (C), followed by incubation with concentrated SN obtained from untransfected or NID1-transfected K562 cells cultured in protein-free medium. NID1 was detected using a goat anti-NID1 Ab followed by a HRP-conjugated anti-goat IgG Ab. Graphs represent absorbance at 405 nm after normalization to background (nonspecific binding of goat anti-NID1 + secondary reagent). Data are medians of triplicates ± interquartile range and are the pooled results of three independent experiments. **** p

    Techniques Used: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture, Binding Assay

    Analysis of NID1 surface expression in HEK293T cell line and in NID1 cell transfectants. (A, B) HEK293T cells were stained with a NID1-specific mAb followed by PE-conjugated anti-mouse IgG1 mAb. Prior to analysis by Amnis ImageStream, cells were incubated with Hoechst 33342 nuclear dye. NID1 expression on individual single cells is shown in (A). Data show representative images from one experiment out of three performed. Histograms in (B) represent total intensity of NID1 immunostaining for HEK293T cells as compared to isotype control. (C) HEK293T, K652, K562-NID1, Bw, and Bw-NID1 cells were stained with a NID1-specific mAb or with NKp44Fc followed by the appropriate isotype-matched PE-conjugated secondary mAb. Samples were analyzed by flow cytometry. Grey profiles represent cells stained with anti-NID1 or with NKp44Fc, while white profiles correspond to isotype control. One representative experiment of four is shown.
    Figure Legend Snippet: Analysis of NID1 surface expression in HEK293T cell line and in NID1 cell transfectants. (A, B) HEK293T cells were stained with a NID1-specific mAb followed by PE-conjugated anti-mouse IgG1 mAb. Prior to analysis by Amnis ImageStream, cells were incubated with Hoechst 33342 nuclear dye. NID1 expression on individual single cells is shown in (A). Data show representative images from one experiment out of three performed. Histograms in (B) represent total intensity of NID1 immunostaining for HEK293T cells as compared to isotype control. (C) HEK293T, K652, K562-NID1, Bw, and Bw-NID1 cells were stained with a NID1-specific mAb or with NKp44Fc followed by the appropriate isotype-matched PE-conjugated secondary mAb. Samples were analyzed by flow cytometry. Grey profiles represent cells stained with anti-NID1 or with NKp44Fc, while white profiles correspond to isotype control. One representative experiment of four is shown.

    Techniques Used: Expressing, Staining, Incubation, Immunostaining, Flow Cytometry, Cytometry

    Analysis of culture supernatant from HEK293T cells by two-dimensional electrophoresis (2-DE). Concentrated HEK293T-SN was analyzed by two-dimensional electrophoresis (2-DE). After blotting, membranes were probed with NKp44Fc (A), NKp46Fc (B), or NKp30Fc (C) followed by HRP-conjugated anti-human IgG mAb. HEK293T-SN-biot was subjected to the same procedure and the membrane was incubated with HRP-conjugated Neutravidin (D). In parallel, HEK293T-SN (600 μg) was separated by 2-DE and proteins were stained with “blue silver” colloidal Coomassie (E). Numbers indicate the spots selected for mass spectrometry analysis; in panels (A), (D), and (E) spot 26 is highlighted with a black circle.
    Figure Legend Snippet: Analysis of culture supernatant from HEK293T cells by two-dimensional electrophoresis (2-DE). Concentrated HEK293T-SN was analyzed by two-dimensional electrophoresis (2-DE). After blotting, membranes were probed with NKp44Fc (A), NKp46Fc (B), or NKp30Fc (C) followed by HRP-conjugated anti-human IgG mAb. HEK293T-SN-biot was subjected to the same procedure and the membrane was incubated with HRP-conjugated Neutravidin (D). In parallel, HEK293T-SN (600 μg) was separated by 2-DE and proteins were stained with “blue silver” colloidal Coomassie (E). Numbers indicate the spots selected for mass spectrometry analysis; in panels (A), (D), and (E) spot 26 is highlighted with a black circle.

    Techniques Used: Electrophoresis, Incubation, Staining, Mass Spectrometry

    29) Product Images from "Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies"

    Article Title: Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02108.x

    Analysis of serum levels of mouse C1q and rabbit IgG at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat anti-Dig-HRP. □, Anti-C1q; , control IgG.
    Figure Legend Snippet: Analysis of serum levels of mouse C1q and rabbit IgG at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat anti-Dig-HRP. □, Anti-C1q; , control IgG.

    Techniques Used: Injection, Mouse Assay, Standard Deviation, Western Blot, Negative Control, SDS Page, Staining

    30) Product Images from "Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies"

    Article Title: Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02108.x

    Analysis of serum levels of mouse C1q and rabbit IgG at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat anti-Dig-HRP. □, Anti-C1q; , control IgG.
    Figure Legend Snippet: Analysis of serum levels of mouse C1q and rabbit IgG at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat anti-Dig-HRP. □, Anti-C1q; , control IgG.

    Techniques Used: Injection, Mouse Assay, Standard Deviation, Western Blot, Negative Control, SDS Page, Staining

    31) Product Images from "A Novel Sensitive Cell-Based Immunoenzymatic Assay for Palytoxin Quantitation in Mussels"

    Article Title: A Novel Sensitive Cell-Based Immunoenzymatic Assay for Palytoxin Quantitation in Mussels

    Journal: Toxins

    doi: 10.3390/toxins10080329

    Palytoxin (PLTX) binding evaluated on a panel of different cell lines, detected by a monoclonal mouse anti-PLTX antibody targeted by horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G. ( A ) Saturation curves of PLTX binding. Box plots showing ( B ) distribution of Kd values and ( C ) maximal bindings obtained by the binding assay for PLTX. Results are expressed as mean ± SE of three experiments performed in triplicate.
    Figure Legend Snippet: Palytoxin (PLTX) binding evaluated on a panel of different cell lines, detected by a monoclonal mouse anti-PLTX antibody targeted by horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G. ( A ) Saturation curves of PLTX binding. Box plots showing ( B ) distribution of Kd values and ( C ) maximal bindings obtained by the binding assay for PLTX. Results are expressed as mean ± SE of three experiments performed in triplicate.

    Techniques Used: Binding Assay

    32) Product Images from "Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic"

    Article Title: Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.760504

    Human TSP-1 recognizes peptide domains within Hic similar to vitronectin and factor H. A and B , pneumococcal proteins were immobilized on microtiter plates (Polysorp TM ; Nunc) in equimolar amounts related to Hic7 (0.5 μg in 100 μl/well). Binding of a constant concentration of hTSP-1 (5 μg/ml) or vitronectin ( Vn ; 1 μg/ml) to immobilized Hic peptides was measured in the presence of increasing molecular ratios of Vn (related to hTSP-1) or hTSP-1 (related to Vn). Glycoprotein binding was detected either with polyclonal mouse anti-hTSP-1 IgG or polyclonal rabbit anti-human Vn antiserum followed by a secondary HRP-coupled goat anti-mouse IgG or HRP-coupled goat anti-rabbit IgG. C and D , pneumococcal Hic2 construct was immobilized on microtiter plates (Maxisorp TM ; Nunc). Binding of a constant concentration of hTSP-1 (2.5 μg/ml) or factor H ( FH , 1 μg/ml) to immobilized Hic2 was measured in the presence of increasing molecular ratios of factor H (related to hTSP-1) or hTSP-1 (related to factor H) using either a polyclonal mouse anti-hTSP-1 IgG or polyclonal goat anti-human FH IgG followed by a secondary HRP-coupled goat anti-mouse IgG or HRP-coupled rabbit anti-goat IgG. E , after immobilization of Hic2 (Maxisorp TM ; Nunc) binding of increasing molecular ratios of hTSP-1 (related to factor H) was measured in presence of a constant concentration of FH (1 μg/ml). Detection of hTSP-1-binding to factor H and/or Hic2 was analyzed as described in C . In A–E , the results are illustrated as mean values ± S.D. of at least three independent experiments. The values of control wells without soluble overlay protein were subtracted from each measured value. *, p
    Figure Legend Snippet: Human TSP-1 recognizes peptide domains within Hic similar to vitronectin and factor H. A and B , pneumococcal proteins were immobilized on microtiter plates (Polysorp TM ; Nunc) in equimolar amounts related to Hic7 (0.5 μg in 100 μl/well). Binding of a constant concentration of hTSP-1 (5 μg/ml) or vitronectin ( Vn ; 1 μg/ml) to immobilized Hic peptides was measured in the presence of increasing molecular ratios of Vn (related to hTSP-1) or hTSP-1 (related to Vn). Glycoprotein binding was detected either with polyclonal mouse anti-hTSP-1 IgG or polyclonal rabbit anti-human Vn antiserum followed by a secondary HRP-coupled goat anti-mouse IgG or HRP-coupled goat anti-rabbit IgG. C and D , pneumococcal Hic2 construct was immobilized on microtiter plates (Maxisorp TM ; Nunc). Binding of a constant concentration of hTSP-1 (2.5 μg/ml) or factor H ( FH , 1 μg/ml) to immobilized Hic2 was measured in the presence of increasing molecular ratios of factor H (related to hTSP-1) or hTSP-1 (related to factor H) using either a polyclonal mouse anti-hTSP-1 IgG or polyclonal goat anti-human FH IgG followed by a secondary HRP-coupled goat anti-mouse IgG or HRP-coupled rabbit anti-goat IgG. E , after immobilization of Hic2 (Maxisorp TM ; Nunc) binding of increasing molecular ratios of hTSP-1 (related to factor H) was measured in presence of a constant concentration of FH (1 μg/ml). Detection of hTSP-1-binding to factor H and/or Hic2 was analyzed as described in C . In A–E , the results are illustrated as mean values ± S.D. of at least three independent experiments. The values of control wells without soluble overlay protein were subtracted from each measured value. *, p

    Techniques Used: Hydrophobic Interaction Chromatography, Binding Assay, Concentration Assay, Construct

    Interaction between Hic2 and BSA. A , microtiter plate (Maxisorp TM ; Nunc) coated with blocking solution containing increasing concentrations of BSA followed by incubation with Hic2. Binding of Hic2 was detected using a polyclonal anti-Hic2 IgG followed by a secondary HRP-coupled goat anti-mouse IgG. The results are illustrated as mean values of one experiment with duplicates. B , interactions of hTSP-1, factor H, and BSA with immobilized Hic2 were analyzed by surface plasmon resonance spectroscopy in manual runs. Therefore, a CM5 sensorchip was coated with native Hic2, and kinetics were conducted in PBS, 0.05% Tween 20 (pH 7.4) with a flow rate of 10 μl/min. The values of the control flow cells were subtracted from each sensorgram. C , binding of Hic2 (10 μg/ml) to immobilized BSA and factor H. Bound Hic was detected using a polyclonal anti-Hic2 IgG and a secondary alkaline phosphatase-coupled goat anti-mouse IgG and NBT/BCIP as substrate.
    Figure Legend Snippet: Interaction between Hic2 and BSA. A , microtiter plate (Maxisorp TM ; Nunc) coated with blocking solution containing increasing concentrations of BSA followed by incubation with Hic2. Binding of Hic2 was detected using a polyclonal anti-Hic2 IgG followed by a secondary HRP-coupled goat anti-mouse IgG. The results are illustrated as mean values of one experiment with duplicates. B , interactions of hTSP-1, factor H, and BSA with immobilized Hic2 were analyzed by surface plasmon resonance spectroscopy in manual runs. Therefore, a CM5 sensorchip was coated with native Hic2, and kinetics were conducted in PBS, 0.05% Tween 20 (pH 7.4) with a flow rate of 10 μl/min. The values of the control flow cells were subtracted from each sensorgram. C , binding of Hic2 (10 μg/ml) to immobilized BSA and factor H. Bound Hic was detected using a polyclonal anti-Hic2 IgG and a secondary alkaline phosphatase-coupled goat anti-mouse IgG and NBT/BCIP as substrate.

    Techniques Used: Blocking Assay, Incubation, Binding Assay, SPR Assay, Spectroscopy, Flow Cytometry, Hydrophobic Interaction Chromatography

    33) Product Images from "Targeting of Voltage-Gated Calcium Channel ?2?-1 Subunit to Lipid Rafts Is Independent from a GPI-Anchoring Motif"

    Article Title: Targeting of Voltage-Gated Calcium Channel ?2?-1 Subunit to Lipid Rafts Is Independent from a GPI-Anchoring Motif

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019802

    Surface and total cellular distribution of PIN-α 2 /δ chimera expressed in COS-7 cells. A. PIN-α 2 /δ. B. PIN-α 2 /δ-PIN TMI . C. PIN-δ c . D. PIN-δ c -PIN TMI . Cells were labelled with anti-HA and Cy5 secondary antibodies using a surface-labelling specific protocol (Methods) and the distribution of surface (red) and total (green, GFP) PIN construct expression determined by fluorescence imaging. Note strong labelling at cell margins for PIN-δ c and PIN-δ c -PIN TMI and highly punctate labelling for PIN-α 2 /δ and PIN-α 2 /δ-PIN TMI . Scale bar 15 µm.
    Figure Legend Snippet: Surface and total cellular distribution of PIN-α 2 /δ chimera expressed in COS-7 cells. A. PIN-α 2 /δ. B. PIN-α 2 /δ-PIN TMI . C. PIN-δ c . D. PIN-δ c -PIN TMI . Cells were labelled with anti-HA and Cy5 secondary antibodies using a surface-labelling specific protocol (Methods) and the distribution of surface (red) and total (green, GFP) PIN construct expression determined by fluorescence imaging. Note strong labelling at cell margins for PIN-δ c and PIN-δ c -PIN TMI and highly punctate labelling for PIN-α 2 /δ and PIN-α 2 /δ-PIN TMI . Scale bar 15 µm.

    Techniques Used: Construct, Expressing, Fluorescence, Imaging

    34) Product Images from "Type I Interferon Impairs Specific Antibody Responses Early during Establishment of LCMV Infection"

    Article Title: Type I Interferon Impairs Specific Antibody Responses Early during Establishment of LCMV Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2016.00564

    LCMV infection impairs the NP-specific Ab response in a T cell-dependent manner . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day (except when indicated otherwise) with an i.p. injection of NP 53 -CGG in alum (A,B,E,F) or NP 40 -FICOLL in PBS (C,D) . (A) NP-specific IgG1 response monitored by ELISA following NP 53 -CGG immunization. (B) Mice were infected as above and immunized with NP 53 -CGG the same day (d0) or 30 days after infection (d30), and IgG1 NP-specific responses were monitored by ELISA on d8 postimmunization. NP-specific IgG3 (C) or IgM (D) responses monitored by ELISA following NP 40 -FICOLL immunization. (E) ELISA plates were coated with NP 4 -BSA or NP 26 -BSA and high affinity Ab responses were measured as a ratio of Abs binding to NP 4 -BSA versus the total anti-NP IgG1 response binding to NP 26 -BSA. (F) Total serum IgG responses following concomitant infection and NP 53 -CGG immunization. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p
    Figure Legend Snippet: LCMV infection impairs the NP-specific Ab response in a T cell-dependent manner . B6 mice (four per group) were infected with LCMV Cl13 (black), LCMV WE (hatched), VSV (gray), or mock infected (white). Mice were immunized the same day (except when indicated otherwise) with an i.p. injection of NP 53 -CGG in alum (A,B,E,F) or NP 40 -FICOLL in PBS (C,D) . (A) NP-specific IgG1 response monitored by ELISA following NP 53 -CGG immunization. (B) Mice were infected as above and immunized with NP 53 -CGG the same day (d0) or 30 days after infection (d30), and IgG1 NP-specific responses were monitored by ELISA on d8 postimmunization. NP-specific IgG3 (C) or IgM (D) responses monitored by ELISA following NP 40 -FICOLL immunization. (E) ELISA plates were coated with NP 4 -BSA or NP 26 -BSA and high affinity Ab responses were measured as a ratio of Abs binding to NP 4 -BSA versus the total anti-NP IgG1 response binding to NP 26 -BSA. (F) Total serum IgG responses following concomitant infection and NP 53 -CGG immunization. Statistical analysis was performed by individual T -tests between experimental groups and the mock-infected group. * p

    Techniques Used: Infection, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay

    35) Product Images from "Glutathione Conjugation at the Blood–CSF Barrier Efficiently Prevents Exposure of the Developing Brain Fluid Environment to Blood-Borne Reactive Electrophilic Substances"

    Article Title: Glutathione Conjugation at the Blood–CSF Barrier Efficiently Prevents Exposure of the Developing Brain Fluid Environment to Blood-Borne Reactive Electrophilic Substances

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2967-17.2018

    Western blot analysis of several GST isoforms in choroid plexus isolated from the lateral and fourth ventricles of the developing rat. Tissue was isolated from 2 P2 ( A , C , D ) and P9 ( B ) animals. A , The GSTμ1/2 isoenzymes are expressed at a higher level in choroid plexuses than in the cerebral cortex and liver. B , Both types of choroid plexuses display similar levels of the GSTμ5 protein, which are higher than in the cerebral cortex and liver. C , The GSTα3 protein is abundant in both types of choroid plexuses relative to the cortical neuropil. D , The GSTπ1 isoenzyme is highly enriched in the choroid plexuses compared with the liver and is also more abundant than in the cerebral cortex. All analyses were repeated on two different sets of samples. CP(LV) and CP(4V), choroid plexuses from the lateral and fourth ventricles, respectively; Cx, Cerebral cortex; Li, liver; MW, molecular weight markers.
    Figure Legend Snippet: Western blot analysis of several GST isoforms in choroid plexus isolated from the lateral and fourth ventricles of the developing rat. Tissue was isolated from 2 P2 ( A , C , D ) and P9 ( B ) animals. A , The GSTμ1/2 isoenzymes are expressed at a higher level in choroid plexuses than in the cerebral cortex and liver. B , Both types of choroid plexuses display similar levels of the GSTμ5 protein, which are higher than in the cerebral cortex and liver. C , The GSTα3 protein is abundant in both types of choroid plexuses relative to the cortical neuropil. D , The GSTπ1 isoenzyme is highly enriched in the choroid plexuses compared with the liver and is also more abundant than in the cerebral cortex. All analyses were repeated on two different sets of samples. CP(LV) and CP(4V), choroid plexuses from the lateral and fourth ventricles, respectively; Cx, Cerebral cortex; Li, liver; MW, molecular weight markers.

    Techniques Used: Western Blot, Isolation, Molecular Weight

    36) Product Images from "Both adhesion to immobilized vitronectin and Fc?RI cross-linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells"

    Article Title: Both adhesion to immobilized vitronectin and Fc?RI cross-linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells

    Journal: Immunology

    doi: 10.1046/j.1365-2567.1999.00883.x

    Mast cell activation via α v ‐integrins and FcεRI results in tyrosine phosphorylation of FAK and stimulates its in vitro kinase activity. (a) Equal numbers of cells were plated on VN PB ‐ or BSA‐treated plates for various times, lysed and immunoprecipitated with anti‐FAK (77) or an isotype‐matched (IgG1) control. Blots were labelled with anti‐phosphotyrosine (PY) and anti‐FAK antibodies. Densitometry scanning of tyrosine phosphorylation of FAK in arbitrary units after normalization against background were: 0 min (869); 1 min (933); 5 min (1130); 15 min (1246); 30 min (1060) and 60 min (1190). FAK protein revealed little change (0 min: 345; 1 min: 328; 5 min: 372; 15 min: 311; 30 min: 349 and 60 min: 431). The average increase in tyrosine phosphorylation of FAK in 15 min compared to 0 min was 1·9 ± 0·62 (mean ± SEM; N = 3) fold. FAK protein was almost the same in each case. (b) Same conditions as above, except that mast cells were incubated with VN sol . Densitometry scanning of tyrosine phosphorylation of FAK was: 0 min (143); 1 min (117); 5 min (76); 15 min (92) and 30 min (65). FAK protein in each case revealed little change (0 min: 157; 1 min: 117; 5 min: 66; 15 min: 143 and 30 min: 144). There was no apparent increase in tyrosine phosphorylation of FAK (0 min: 158 ± 38; 1 min: 134 ± 36; 5 min: 117 ± 37; 15 min: 130 ± 37; 30 min: 113 ± 36; mean ± SEM; N = 4). FAK proteins remained similar. (c) Sensitized non‐adherent mast cells were activated by addition of specific antigen (DNP‐BSA). Densitometry scanning of tyrosine phosphorylation of FAK was: 0 min (82); 1 min (128); 5 min (233); 15 min (163) and 30 min (112). FAK protein revealed little change (0 min: 356; 1 min: 367; 5 min: 335; 15 min: 364 and 30 min: 359). The average increase in tyrosine phosphorylation of FAK at 5 min compared to 0 min was 3·15 ± 0·35 (mean ± SEM; N = 3)‐fold. FAK protein was similar in each case. (d) Mast cells were activated by IgE receptor cross‐linking (5 min) in the presence of VN PB (15 min) and independently by IgE receptor cross‐linking (5 min) and VN PB (15 min). Tyrosine phosphorylation of FAK by densitometry in lanes 1, 2, 3 and 4 was 262, 932, 1595 and 834, respectively. Scanning of FAK proteins in the respective lanes was 546, 697, 697 and 482. In another experiment, tyrosine phosphorylation was increased sixfold by both stimuli together and approximately threefold by each stimulus alone compared to baseline. FAK protein was similar in each case. (e) Mast cells activated by VN PB or by FcɛRI cross‐linking were immunoprecipitated and in vitro kinase activity of FAK was measured (see Materials and methods). IP equals immunoprecipitating antibody and IB equals immunoblotting antibody.
    Figure Legend Snippet: Mast cell activation via α v ‐integrins and FcεRI results in tyrosine phosphorylation of FAK and stimulates its in vitro kinase activity. (a) Equal numbers of cells were plated on VN PB ‐ or BSA‐treated plates for various times, lysed and immunoprecipitated with anti‐FAK (77) or an isotype‐matched (IgG1) control. Blots were labelled with anti‐phosphotyrosine (PY) and anti‐FAK antibodies. Densitometry scanning of tyrosine phosphorylation of FAK in arbitrary units after normalization against background were: 0 min (869); 1 min (933); 5 min (1130); 15 min (1246); 30 min (1060) and 60 min (1190). FAK protein revealed little change (0 min: 345; 1 min: 328; 5 min: 372; 15 min: 311; 30 min: 349 and 60 min: 431). The average increase in tyrosine phosphorylation of FAK in 15 min compared to 0 min was 1·9 ± 0·62 (mean ± SEM; N = 3) fold. FAK protein was almost the same in each case. (b) Same conditions as above, except that mast cells were incubated with VN sol . Densitometry scanning of tyrosine phosphorylation of FAK was: 0 min (143); 1 min (117); 5 min (76); 15 min (92) and 30 min (65). FAK protein in each case revealed little change (0 min: 157; 1 min: 117; 5 min: 66; 15 min: 143 and 30 min: 144). There was no apparent increase in tyrosine phosphorylation of FAK (0 min: 158 ± 38; 1 min: 134 ± 36; 5 min: 117 ± 37; 15 min: 130 ± 37; 30 min: 113 ± 36; mean ± SEM; N = 4). FAK proteins remained similar. (c) Sensitized non‐adherent mast cells were activated by addition of specific antigen (DNP‐BSA). Densitometry scanning of tyrosine phosphorylation of FAK was: 0 min (82); 1 min (128); 5 min (233); 15 min (163) and 30 min (112). FAK protein revealed little change (0 min: 356; 1 min: 367; 5 min: 335; 15 min: 364 and 30 min: 359). The average increase in tyrosine phosphorylation of FAK at 5 min compared to 0 min was 3·15 ± 0·35 (mean ± SEM; N = 3)‐fold. FAK protein was similar in each case. (d) Mast cells were activated by IgE receptor cross‐linking (5 min) in the presence of VN PB (15 min) and independently by IgE receptor cross‐linking (5 min) and VN PB (15 min). Tyrosine phosphorylation of FAK by densitometry in lanes 1, 2, 3 and 4 was 262, 932, 1595 and 834, respectively. Scanning of FAK proteins in the respective lanes was 546, 697, 697 and 482. In another experiment, tyrosine phosphorylation was increased sixfold by both stimuli together and approximately threefold by each stimulus alone compared to baseline. FAK protein was similar in each case. (e) Mast cells activated by VN PB or by FcɛRI cross‐linking were immunoprecipitated and in vitro kinase activity of FAK was measured (see Materials and methods). IP equals immunoprecipitating antibody and IB equals immunoblotting antibody.

    Techniques Used: Activation Assay, In Vitro, Activity Assay, Immunoprecipitation, Incubation

    37) Product Images from "Azadirachta indica A. Juss Ameliorates Mouse Hepatitis Virus-Induced Neuroinflammatory Demyelination by Modulating Cell-to-Cell Fusion in an Experimental Animal Model of Multiple Sclerosis"

    Article Title: Azadirachta indica A. Juss Ameliorates Mouse Hepatitis Virus-Induced Neuroinflammatory Demyelination by Modulating Cell-to-Cell Fusion in an Experimental Animal Model of Multiple Sclerosis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2020.00116

    Intracranial infection in mice with RSA59, preincubated with NBE, restricted viral replication at day 6 p.i., implying a direct anti-viral property in vivo . Day-timeline of intracranial infection (i.c.) of C57BL/6 mice and sacrifice (A) . Mice were infected with RSA59 (20,000 PFU; OV), NBE-preincubated (25 mg/kg B.W.) RSA59 (T 25), and RSA59 preincubated with DMSO (V 25). The comparative effect of NBE on body weight change was shown upon i.c. inoculation with NBE-preincubated RSA59 into C57BL/6 mice brain. Significant changes were observed at day 2 and 3 p.i. in T 25 mice compared with V 25 mice (B) . Tissue lysates from the brain and spinal cord from OV, T and V mice were subjected to comparative viral plaque assay on confluent monolayers of L2 cells at day 6 p.i. Titers were expressed as log 10 PFU/gm of tissue. T 25 revealed potential downregulation in RSA59 replication compared to V in the brain (C) and spinal cord (D) . The relative expression of viral N and S transcripts upon NBE treatment was determined in the brain tissues at day 6 p.i using qRT-PCR analysis. Results were normalized to GAPDH, and compared with OV. There is significant downregulation in N (E) and S (F) gene expression at the mRNA level during the acute infection stage. Similarly, mouse whole brain and liver lysates at day 6 p.i. were subjected to immunoblot analysis with Anti-N (Nucleocapsid) antibody at 1:50 dilution (G) . Significant downregulation of viral N expression at the protein level was observed in both brain (H) and liver (I) tissues. Results were normalized to γ-Actin, and compared with OV. Data represent mean ± SEM in scatter plots and statistical significance was determined by unpaired student’s t -test and RM Two-way ANOVA followed by Sidak’s multiple comparison test; * p
    Figure Legend Snippet: Intracranial infection in mice with RSA59, preincubated with NBE, restricted viral replication at day 6 p.i., implying a direct anti-viral property in vivo . Day-timeline of intracranial infection (i.c.) of C57BL/6 mice and sacrifice (A) . Mice were infected with RSA59 (20,000 PFU; OV), NBE-preincubated (25 mg/kg B.W.) RSA59 (T 25), and RSA59 preincubated with DMSO (V 25). The comparative effect of NBE on body weight change was shown upon i.c. inoculation with NBE-preincubated RSA59 into C57BL/6 mice brain. Significant changes were observed at day 2 and 3 p.i. in T 25 mice compared with V 25 mice (B) . Tissue lysates from the brain and spinal cord from OV, T and V mice were subjected to comparative viral plaque assay on confluent monolayers of L2 cells at day 6 p.i. Titers were expressed as log 10 PFU/gm of tissue. T 25 revealed potential downregulation in RSA59 replication compared to V in the brain (C) and spinal cord (D) . The relative expression of viral N and S transcripts upon NBE treatment was determined in the brain tissues at day 6 p.i using qRT-PCR analysis. Results were normalized to GAPDH, and compared with OV. There is significant downregulation in N (E) and S (F) gene expression at the mRNA level during the acute infection stage. Similarly, mouse whole brain and liver lysates at day 6 p.i. were subjected to immunoblot analysis with Anti-N (Nucleocapsid) antibody at 1:50 dilution (G) . Significant downregulation of viral N expression at the protein level was observed in both brain (H) and liver (I) tissues. Results were normalized to γ-Actin, and compared with OV. Data represent mean ± SEM in scatter plots and statistical significance was determined by unpaired student’s t -test and RM Two-way ANOVA followed by Sidak’s multiple comparison test; * p

    Techniques Used: Infection, Mouse Assay, In Vivo, Viral Plaque Assay, Expressing, Quantitative RT-PCR

    Intraperitoneal administration of NBE exhibited a significant reduction in viral Nucleocapsid protein expression. Day-timeline of intracranial infection (i.c.) following intraperitoneal (i.p.) administration of NBE into C57BL/6 mice and sacrifice (A) . Mice were infected with MHV-A59 (2,000 PFU; OV) and NBE (25 mg/kg B.W.) was administered i.p. following the MHV-A59 infection (T). Mouse whole brain and liver lysates at day 5 p.i. and day 7 p.i. were subjected to immunoblot analysis with Anti-N (Nucleocapsid) antibody at 1:50 dilution (B) . Results were normalized to γ-Actin, and compared with OV. At the acute stage of infection, NBE significantly decreased viral “N” protein expression in the brain (B,C,E) and spinal cord (B,D,F) proteins at both 5 and 7 days p.i. followed by i.p. NBE administration. Data were represented as mean ± SEM and statistical significance was determined by unpaired student’s t -test analysis; **** p
    Figure Legend Snippet: Intraperitoneal administration of NBE exhibited a significant reduction in viral Nucleocapsid protein expression. Day-timeline of intracranial infection (i.c.) following intraperitoneal (i.p.) administration of NBE into C57BL/6 mice and sacrifice (A) . Mice were infected with MHV-A59 (2,000 PFU; OV) and NBE (25 mg/kg B.W.) was administered i.p. following the MHV-A59 infection (T). Mouse whole brain and liver lysates at day 5 p.i. and day 7 p.i. were subjected to immunoblot analysis with Anti-N (Nucleocapsid) antibody at 1:50 dilution (B) . Results were normalized to γ-Actin, and compared with OV. At the acute stage of infection, NBE significantly decreased viral “N” protein expression in the brain (B,C,E) and spinal cord (B,D,F) proteins at both 5 and 7 days p.i. followed by i.p. NBE administration. Data were represented as mean ± SEM and statistical significance was determined by unpaired student’s t -test analysis; **** p

    Techniques Used: Expressing, Infection, Mouse Assay

    38) Product Images from "Azadirachta indica A. Juss Ameliorates Mouse Hepatitis Virus-Induced Neuroinflammatory Demyelination by Modulating Cell-to-Cell Fusion in an Experimental Animal Model of Multiple Sclerosis"

    Article Title: Azadirachta indica A. Juss Ameliorates Mouse Hepatitis Virus-Induced Neuroinflammatory Demyelination by Modulating Cell-to-Cell Fusion in an Experimental Animal Model of Multiple Sclerosis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2020.00116

    Intracranial infection in mice with RSA59, preincubated with NBE, restricted viral replication at day 6 p.i., implying a direct anti-viral property in vivo . Day-timeline of intracranial infection (i.c.) of C57BL/6 mice and sacrifice (A) . Mice were infected with RSA59 (20,000 PFU; OV), NBE-preincubated (25 mg/kg B.W.) RSA59 (T 25), and RSA59 preincubated with DMSO (V 25). The comparative effect of NBE on body weight change was shown upon i.c. inoculation with NBE-preincubated RSA59 into C57BL/6 mice brain. Significant changes were observed at day 2 and 3 p.i. in T 25 mice compared with V 25 mice (B) . Tissue lysates from the brain and spinal cord from OV, T and V mice were subjected to comparative viral plaque assay on confluent monolayers of L2 cells at day 6 p.i. Titers were expressed as log 10 PFU/gm of tissue. T 25 revealed potential downregulation in RSA59 replication compared to V in the brain (C) and spinal cord (D) . The relative expression of viral N and S transcripts upon NBE treatment was determined in the brain tissues at day 6 p.i using qRT-PCR analysis. Results were normalized to GAPDH, and compared with OV. There is significant downregulation in N (E) and S (F) gene expression at the mRNA level during the acute infection stage. Similarly, mouse whole brain and liver lysates at day 6 p.i. were subjected to immunoblot analysis with Anti-N (Nucleocapsid) antibody at 1:50 dilution (G) . Significant downregulation of viral N expression at the protein level was observed in both brain (H) and liver (I) tissues. Results were normalized to γ-Actin, and compared with OV. Data represent mean ± SEM in scatter plots and statistical significance was determined by unpaired student’s t -test and RM Two-way ANOVA followed by Sidak’s multiple comparison test; * p
    Figure Legend Snippet: Intracranial infection in mice with RSA59, preincubated with NBE, restricted viral replication at day 6 p.i., implying a direct anti-viral property in vivo . Day-timeline of intracranial infection (i.c.) of C57BL/6 mice and sacrifice (A) . Mice were infected with RSA59 (20,000 PFU; OV), NBE-preincubated (25 mg/kg B.W.) RSA59 (T 25), and RSA59 preincubated with DMSO (V 25). The comparative effect of NBE on body weight change was shown upon i.c. inoculation with NBE-preincubated RSA59 into C57BL/6 mice brain. Significant changes were observed at day 2 and 3 p.i. in T 25 mice compared with V 25 mice (B) . Tissue lysates from the brain and spinal cord from OV, T and V mice were subjected to comparative viral plaque assay on confluent monolayers of L2 cells at day 6 p.i. Titers were expressed as log 10 PFU/gm of tissue. T 25 revealed potential downregulation in RSA59 replication compared to V in the brain (C) and spinal cord (D) . The relative expression of viral N and S transcripts upon NBE treatment was determined in the brain tissues at day 6 p.i using qRT-PCR analysis. Results were normalized to GAPDH, and compared with OV. There is significant downregulation in N (E) and S (F) gene expression at the mRNA level during the acute infection stage. Similarly, mouse whole brain and liver lysates at day 6 p.i. were subjected to immunoblot analysis with Anti-N (Nucleocapsid) antibody at 1:50 dilution (G) . Significant downregulation of viral N expression at the protein level was observed in both brain (H) and liver (I) tissues. Results were normalized to γ-Actin, and compared with OV. Data represent mean ± SEM in scatter plots and statistical significance was determined by unpaired student’s t -test and RM Two-way ANOVA followed by Sidak’s multiple comparison test; * p

    Techniques Used: Infection, Mouse Assay, In Vivo, Viral Plaque Assay, Expressing, Quantitative RT-PCR

    Intraperitoneal administration of NBE exhibited a significant reduction in viral Nucleocapsid protein expression. Day-timeline of intracranial infection (i.c.) following intraperitoneal (i.p.) administration of NBE into C57BL/6 mice and sacrifice (A) . Mice were infected with MHV-A59 (2,000 PFU; OV) and NBE (25 mg/kg B.W.) was administered i.p. following the MHV-A59 infection (T). Mouse whole brain and liver lysates at day 5 p.i. and day 7 p.i. were subjected to immunoblot analysis with Anti-N (Nucleocapsid) antibody at 1:50 dilution (B) . Results were normalized to γ-Actin, and compared with OV. At the acute stage of infection, NBE significantly decreased viral “N” protein expression in the brain (B,C,E) and spinal cord (B,D,F) proteins at both 5 and 7 days p.i. followed by i.p. NBE administration. Data were represented as mean ± SEM and statistical significance was determined by unpaired student’s t -test analysis; **** p
    Figure Legend Snippet: Intraperitoneal administration of NBE exhibited a significant reduction in viral Nucleocapsid protein expression. Day-timeline of intracranial infection (i.c.) following intraperitoneal (i.p.) administration of NBE into C57BL/6 mice and sacrifice (A) . Mice were infected with MHV-A59 (2,000 PFU; OV) and NBE (25 mg/kg B.W.) was administered i.p. following the MHV-A59 infection (T). Mouse whole brain and liver lysates at day 5 p.i. and day 7 p.i. were subjected to immunoblot analysis with Anti-N (Nucleocapsid) antibody at 1:50 dilution (B) . Results were normalized to γ-Actin, and compared with OV. At the acute stage of infection, NBE significantly decreased viral “N” protein expression in the brain (B,C,E) and spinal cord (B,D,F) proteins at both 5 and 7 days p.i. followed by i.p. NBE administration. Data were represented as mean ± SEM and statistical significance was determined by unpaired student’s t -test analysis; **** p

    Techniques Used: Expressing, Infection, Mouse Assay

    39) Product Images from "BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian embryogenesis"

    Article Title: BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian embryogenesis

    Journal: bioRxiv

    doi: 10.1101/686758

    Endogenous BMP4 co-immunoprecipitates with BMP7. Antibodies specific for Flag-epitope tag were used to immunoprecipitate (IP) proteins from E11.5 Bmp7 +/+ , Bmp7 R-GFlag/+ or Bmp7 Flag/+ lysates. Immunoblots of IPs or total protein (input) were probed with antibodies specific for BMP4 or Flag as indicated below each panel. The position of precursor proteins and cleaved mature ligand is indicated on the right. Arrows indicates band corresponding to IgG heavy or light chains, asterisks mark non-specific bands. Results were reproduced in two independent experiments.
    Figure Legend Snippet: Endogenous BMP4 co-immunoprecipitates with BMP7. Antibodies specific for Flag-epitope tag were used to immunoprecipitate (IP) proteins from E11.5 Bmp7 +/+ , Bmp7 R-GFlag/+ or Bmp7 Flag/+ lysates. Immunoblots of IPs or total protein (input) were probed with antibodies specific for BMP4 or Flag as indicated below each panel. The position of precursor proteins and cleaved mature ligand is indicated on the right. Arrows indicates band corresponding to IgG heavy or light chains, asterisks mark non-specific bands. Results were reproduced in two independent experiments.

    Techniques Used: FLAG-tag, Western Blot

    40) Product Images from "Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies"

    Article Title: Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02108.x

    Analysis of serum levels of mouse C1q and rabbit IgG at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat anti-Dig-HRP. □, Anti-C1q; , control IgG.
    Figure Legend Snippet: Analysis of serum levels of mouse C1q and rabbit IgG at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat anti-Dig-HRP. □, Anti-C1q; , control IgG.

    Techniques Used: Injection, Mouse Assay, Standard Deviation, Western Blot, Negative Control, SDS Page, Staining

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    Generated:

    Article Title: Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model
    Article Snippet: .. A 1:2000 dilution of HRP-conjugated goat antimouse IgG, IgM, IgG1, IgG2b, IgG2c, or IgG3 (Jackson ImmunoResearch Laboratory) in 0.1% BSA/PBS (100μL) is added to the wells, respectively, to determine the subtypes of antibodies generated. ..

    other:

    Article Title: Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model
    Article Snippet: BSA-Tn (1mg/mL) NaHCO3 /Na2 CO3 buffer (0.05 M , pH 9.6): Na2 CO3 (159mg), NaHCO3 (293mg), NaN3 (20mg), H2 O (100mL) PBS/0.5% Tween-20 (PBST) 1% (w/v) BSA Horseradish peroxidase (HRP)-conjugated goat antimouse IgG, IgM, IgG1, IgG2b, IgG2c, or IgG3 (Jackson ImmunoResearch Laboratory) 3,3′,5,5′-Tetramethylbenzidine (TMB) 0.5 M H2 SO4 Citric acid buffer: Na2 HPO4 (7.5g), citric acid monohydrate (4.57g) in distilled water (1L)

    Incubation:

    Article Title: IRF9 and STAT1 are required for IgG autoantibody production and B cell expression of TLR7 in mice
    Article Snippet: .. Wells were incubated with mouse sera diluted 1:250 in PBS containing 3% FBS and 0.05% Tween-20 followed by incubation with HRP-conjugated goat anti-mouse IgG, IgM, IgG1, IgG2a, IgG2b, or IgG3 secondary antibody (Jackson ImmunoResearch Laboratories) at a dilution of 1:5,000. .. Tetramethylbenzidine substrate (Pierce) was added, and OD values were determined at 450 nm.

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    Jackson Immuno horseradish peroxidase hrp conjugated anti mouse igg secondary antibody
    A high-throughput assay (EJIPT) utilizing EJC binding to spliced mRNAs to measure splicing in vitro . Splicing reaction mixtures containing biotin-labeled pre-mRNA, 20 μM each compound, splicing extract, and 0.5 mM ATP are assembled by liquid handler in 384-well plates and incubated for 1.5 h at 30°C. Reaction mixtures are then transferred to a NeutrAvidin-coated plate, which captures biotin-labeled RNAs/RNPs postsplicing. Anti-eIF4AIII (3F1) antibody is added to detect splicing-dependent EJC formation on spliced mRNAs. Following a washing step, incubation with an <t>HRP-conjugated</t> anti-mouse <t>IgG</t> secondary antibody, and then addition of chemiluminescent substrate, signals representing eIF4AIII/mRNA complexes are obtained by an automatic plate reader.
    Horseradish Peroxidase Hrp Conjugated Anti Mouse Igg Secondary Antibody, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno hrp conjugated goat anti mouse
    JK-6L cells show enhanced protein accumulation in the presence of bortezomib and verapamil. (A) Western blot analysis of detergent-soluble and detergent-insoluble fractions of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hours. Immunoblot analysiswas done using an anti-ubiquitin antibody. To detect free ubiquitin, a denser gel was used. One representative immunoblot of two independent experiments is shown. (B) Western blot analysis of total cell lysates fromJK-6L cells treated with 10 nM bortezomib and/or 70 µMverapamil for 16 hours. Immunoblot analysis was done using <t>HRP-conjugated</t> <t>anti-IgG</t> antibody. One representative immunoblot of two independent experiments is shown. (C) IgG concentration in the supernatant of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hourswas determined by ELISA. The absorbance (OD)wasmeasured using a spectrophotometer. Mean values and SDwere calculated fromhexaplicates. Data represent one of two independently done experiments. Student's t -test for unpaired heteroscedastic samples was used for statistical analysis. # P
    Hrp Conjugated Goat Anti Mouse, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno donkey anti goat igg horseradish peroxidase conjugated secondary antibody
    100-fold less RABV-ED51-mBAFF is needed to induce comparable immunity in mice immunized with RABV. C57BL/6J mice were immunized i.m. with 10 3 or 10 5 ffu/mouse of RABV or RABV-ED51-mBAFF, or <t>PBS</t> as a negative control. Blood was collected on days 5, 7 and 10 post-immunization as a source of serum. Four three-fold serial dilutions of sera were analyzed by ELISA to determine anti-RABV G IgM (A and B) or <t>IgG</t> (C and D) antibody titers and presented as OD 490 of the reciprocal serial dilution. For comparison, sera from PBS-immunized mice were tested in parallel. Statistical difference in antibody titers by ELISA between RABV- and RABV-ED51-mBAFF-immunized mice was determined using an unpaired, two-tailed t test and data is presented as the mean ± SEM. *p≤0.05; **p ≤ 0.01; ***p≤0.001; ****p≤0.0001; N = 10 mice/group). (ffu = focus forming units; OD = optical density).
    Donkey Anti Goat Igg Horseradish Peroxidase Conjugated Secondary Antibody, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno goat antirabbit igg conjugated to hrp
    Analysis of serum levels of mouse C1q and rabbit <t>IgG</t> at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat <t>anti-Dig-HRP.</t> □, Anti-C1q; , control IgG.
    Goat Antirabbit Igg Conjugated To Hrp, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A high-throughput assay (EJIPT) utilizing EJC binding to spliced mRNAs to measure splicing in vitro . Splicing reaction mixtures containing biotin-labeled pre-mRNA, 20 μM each compound, splicing extract, and 0.5 mM ATP are assembled by liquid handler in 384-well plates and incubated for 1.5 h at 30°C. Reaction mixtures are then transferred to a NeutrAvidin-coated plate, which captures biotin-labeled RNAs/RNPs postsplicing. Anti-eIF4AIII (3F1) antibody is added to detect splicing-dependent EJC formation on spliced mRNAs. Following a washing step, incubation with an HRP-conjugated anti-mouse IgG secondary antibody, and then addition of chemiluminescent substrate, signals representing eIF4AIII/mRNA complexes are obtained by an automatic plate reader.

    Journal: Molecular and Cellular Biology

    Article Title: A Quantitative High-Throughput In Vitro Splicing Assay Identifies Inhibitors of Spliceosome Catalysis

    doi: 10.1128/MCB.05788-11

    Figure Lengend Snippet: A high-throughput assay (EJIPT) utilizing EJC binding to spliced mRNAs to measure splicing in vitro . Splicing reaction mixtures containing biotin-labeled pre-mRNA, 20 μM each compound, splicing extract, and 0.5 mM ATP are assembled by liquid handler in 384-well plates and incubated for 1.5 h at 30°C. Reaction mixtures are then transferred to a NeutrAvidin-coated plate, which captures biotin-labeled RNAs/RNPs postsplicing. Anti-eIF4AIII (3F1) antibody is added to detect splicing-dependent EJC formation on spliced mRNAs. Following a washing step, incubation with an HRP-conjugated anti-mouse IgG secondary antibody, and then addition of chemiluminescent substrate, signals representing eIF4AIII/mRNA complexes are obtained by an automatic plate reader.

    Article Snippet: Plates were washed in six cycles with HNT buffer on the ELx405, and then a horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (Jackson Immunologicals) diluted 1:10,000 in HNT buffer was added and incubated for 1 h. After a final series of identical washes, 50 μl of Super Signal ELISA (enzyme-linked immunosorbent assay) Fempto chemiluminescent substrate (Pierce) was added, and luminescence signals were measured on a Perkin Elmer Envision reader.

    Techniques: High Throughput Screening Assay, Binding Assay, In Vitro, Labeling, Incubation

    JK-6L cells show enhanced protein accumulation in the presence of bortezomib and verapamil. (A) Western blot analysis of detergent-soluble and detergent-insoluble fractions of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hours. Immunoblot analysiswas done using an anti-ubiquitin antibody. To detect free ubiquitin, a denser gel was used. One representative immunoblot of two independent experiments is shown. (B) Western blot analysis of total cell lysates fromJK-6L cells treated with 10 nM bortezomib and/or 70 µMverapamil for 16 hours. Immunoblot analysis was done using HRP-conjugated anti-IgG antibody. One representative immunoblot of two independent experiments is shown. (C) IgG concentration in the supernatant of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hourswas determined by ELISA. The absorbance (OD)wasmeasured using a spectrophotometer. Mean values and SDwere calculated fromhexaplicates. Data represent one of two independently done experiments. Student's t -test for unpaired heteroscedastic samples was used for statistical analysis. # P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Calcium Channel Blocker Verapamil Enhances Endoplasmic Reticulum Stress and Cell Death Induced by Proteasome Inhibition in Myeloma Cells 1Calcium Channel Blocker Verapamil Enhances Endoplasmic Reticulum Stress and Cell Death Induced by Proteasome Inhibition in Myeloma Cells 1 2

    doi:

    Figure Lengend Snippet: JK-6L cells show enhanced protein accumulation in the presence of bortezomib and verapamil. (A) Western blot analysis of detergent-soluble and detergent-insoluble fractions of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hours. Immunoblot analysiswas done using an anti-ubiquitin antibody. To detect free ubiquitin, a denser gel was used. One representative immunoblot of two independent experiments is shown. (B) Western blot analysis of total cell lysates fromJK-6L cells treated with 10 nM bortezomib and/or 70 µMverapamil for 16 hours. Immunoblot analysis was done using HRP-conjugated anti-IgG antibody. One representative immunoblot of two independent experiments is shown. (C) IgG concentration in the supernatant of JK-6L cells treated with 10 nM bortezomib and/or 70 µ M verapamil for 16 hourswas determined by ELISA. The absorbance (OD)wasmeasured using a spectrophotometer. Mean values and SDwere calculated fromhexaplicates. Data represent one of two independently done experiments. Student's t -test for unpaired heteroscedastic samples was used for statistical analysis. # P

    Article Snippet: As secondary antibodies, we used HRP-conjugated goat anti-mouse immunoglobulinG(IgG), goat antirabbit IgG (Jackson Immunoresearch Laboratories, Inc, West Grove, PA), and donkey antigoat IgG (Santa Cruz Biotechnology).

    Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Spectrophotometry

    100-fold less RABV-ED51-mBAFF is needed to induce comparable immunity in mice immunized with RABV. C57BL/6J mice were immunized i.m. with 10 3 or 10 5 ffu/mouse of RABV or RABV-ED51-mBAFF, or PBS as a negative control. Blood was collected on days 5, 7 and 10 post-immunization as a source of serum. Four three-fold serial dilutions of sera were analyzed by ELISA to determine anti-RABV G IgM (A and B) or IgG (C and D) antibody titers and presented as OD 490 of the reciprocal serial dilution. For comparison, sera from PBS-immunized mice were tested in parallel. Statistical difference in antibody titers by ELISA between RABV- and RABV-ED51-mBAFF-immunized mice was determined using an unpaired, two-tailed t test and data is presented as the mean ± SEM. *p≤0.05; **p ≤ 0.01; ***p≤0.001; ****p≤0.0001; N = 10 mice/group). (ffu = focus forming units; OD = optical density).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Incorporating B cell activating factor (BAFF) into the membrane of rabies virus (RABV) particles improves the speed and magnitude of vaccine-induced antibody responses

    doi: 10.1371/journal.pntd.0007800

    Figure Lengend Snippet: 100-fold less RABV-ED51-mBAFF is needed to induce comparable immunity in mice immunized with RABV. C57BL/6J mice were immunized i.m. with 10 3 or 10 5 ffu/mouse of RABV or RABV-ED51-mBAFF, or PBS as a negative control. Blood was collected on days 5, 7 and 10 post-immunization as a source of serum. Four three-fold serial dilutions of sera were analyzed by ELISA to determine anti-RABV G IgM (A and B) or IgG (C and D) antibody titers and presented as OD 490 of the reciprocal serial dilution. For comparison, sera from PBS-immunized mice were tested in parallel. Statistical difference in antibody titers by ELISA between RABV- and RABV-ED51-mBAFF-immunized mice was determined using an unpaired, two-tailed t test and data is presented as the mean ± SEM. *p≤0.05; **p ≤ 0.01; ***p≤0.001; ****p≤0.0001; N = 10 mice/group). (ffu = focus forming units; OD = optical density).

    Article Snippet: Membrane was probed for one hour with polyclonal goat IgG anti-murine BAFF primary antibody (AF2106; R & D Systems) at a dilution of 1:2,000 in PBS-0.05% Tween-20 (PBS-T), washed 3 times with PBS-T, then incubated for one hour with donkey anti-goat IgG horseradish peroxidase-conjugated secondary antibody (Jackson Immuno) diluted 1:30,000 in PBS-T.

    Techniques: Mouse Assay, Negative Control, Enzyme-linked Immunosorbent Assay, Serial Dilution, Two Tailed Test

    Analysis of serum levels of mouse C1q and rabbit IgG at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat anti-Dig-HRP. □, Anti-C1q; , control IgG.

    Journal: Clinical and Experimental Immunology

    Article Title: Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

    doi: 10.1046/j.1365-2249.2003.02108.x

    Figure Lengend Snippet: Analysis of serum levels of mouse C1q and rabbit IgG at 24 h after injection. Assessment of serum levels of C1q (a) and rabbit IgG (c) of control mice and in sera of mice sacrificed 24 h after injection with anti-C1q or IgG. The mean and standard deviation are shown ( n = 5 mice per group). (b) Western blot analysis of mouse sera, obtained 24 h after injection, of mice injected with anti-C1q or control rabbit IgG with NMS as a positive and C1q -/- as a negative control. Total serum was separated on a 10% SDS-PAGE gel under reducing conditions, blotted onto nitrocellulose and stained with Dig-conjugated rabbit antimouse C1q followed by goat anti-Dig-HRP. □, Anti-C1q; , control IgG.

    Article Snippet: After washing, rabbit IgG binding was detected using goat antirabbit IgG conjugated to HRP (Jackson) in PBS-T-BSA containing 1 m NaCl.

    Techniques: Injection, Mouse Assay, Standard Deviation, Western Blot, Negative Control, SDS Page, Staining