horseradish peroxidase conjugated secondary antibody  (GE Healthcare)

 
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    GE Healthcare horseradish peroxidase conjugated secondary antibody
    A) Induced SR-BI expression in HEK293 cells. Expression of SR-BI was induced by different concentrations of Doxycycline as indicated on the figure. Western blot analysis was performed with anti-SR-BI <t>antibody</t> (Novus Biologicals, NB400-113). <t>Horseradish</t> <t>peroxidase-conjugated</t> <t>secondary</t> antibody and the enhanced chemiluminescent substrate system (Amersham) were used. B) Bodipy-CE uptake is dependent on the expression level of SR-BI. After Doxycycline induction, HEK293[pTRE-tight-SR-BI] cells were incubated with 10 µg/mL DiO-HDL. The selective uptake was determined as described in Experimental Procedures. Data are presented as mean relative fluorescence units from triplicate or duplicate wells. Error bars represent ±SD.
    Horseradish Peroxidase Conjugated Secondary Antibody, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 614 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase conjugated secondary antibody/product/GE Healthcare
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    92/100 stars

    Images

    1) Product Images from "An apoA-I mimetic peptide facilitates off-loading cholesterol from HDL to liver cells through scavenger receptor BI"

    Article Title: An apoA-I mimetic peptide facilitates off-loading cholesterol from HDL to liver cells through scavenger receptor BI

    Journal: International Journal of Biological Sciences

    doi:

    A) Induced SR-BI expression in HEK293 cells. Expression of SR-BI was induced by different concentrations of Doxycycline as indicated on the figure. Western blot analysis was performed with anti-SR-BI antibody (Novus Biologicals, NB400-113). Horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescent substrate system (Amersham) were used. B) Bodipy-CE uptake is dependent on the expression level of SR-BI. After Doxycycline induction, HEK293[pTRE-tight-SR-BI] cells were incubated with 10 µg/mL DiO-HDL. The selective uptake was determined as described in Experimental Procedures. Data are presented as mean relative fluorescence units from triplicate or duplicate wells. Error bars represent ±SD.
    Figure Legend Snippet: A) Induced SR-BI expression in HEK293 cells. Expression of SR-BI was induced by different concentrations of Doxycycline as indicated on the figure. Western blot analysis was performed with anti-SR-BI antibody (Novus Biologicals, NB400-113). Horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescent substrate system (Amersham) were used. B) Bodipy-CE uptake is dependent on the expression level of SR-BI. After Doxycycline induction, HEK293[pTRE-tight-SR-BI] cells were incubated with 10 µg/mL DiO-HDL. The selective uptake was determined as described in Experimental Procedures. Data are presented as mean relative fluorescence units from triplicate or duplicate wells. Error bars represent ±SD.

    Techniques Used: Expressing, Western Blot, Incubation, Fluorescence

    2) Product Images from "Serotonylation of Vascular Proteins Important to Contraction"

    Article Title: Serotonylation of Vascular Proteins Important to Contraction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005682

    α-actin is serotonylated in aortic smooth muscle cells and inhibition of TG activity reduces aortic contraction to 5-HT. A. Immunoprecipitation of smooth muscle α-actin from rat aortic homogenates exposed to 5-HT-biotin in a standard transglutaminase reaction. Blots were developed using a streptavidin secondary (top), or exposed to a primary antibody against α-actin (bottom) and developed using standard horseradish peroxidase secondary antibody. Representative of N = 6 different experiments. B. Effect of vehicle (filled symbol) and cystamine (0.1–1 mM; open symbol) on 5-HT (top) and KCl (bottom)-induced contraction in isolated rat aorta. * indicates statistical difference from vehicle-incubated values. Points and vertical lines represent means±SEM for number of animals in parentheses.
    Figure Legend Snippet: α-actin is serotonylated in aortic smooth muscle cells and inhibition of TG activity reduces aortic contraction to 5-HT. A. Immunoprecipitation of smooth muscle α-actin from rat aortic homogenates exposed to 5-HT-biotin in a standard transglutaminase reaction. Blots were developed using a streptavidin secondary (top), or exposed to a primary antibody against α-actin (bottom) and developed using standard horseradish peroxidase secondary antibody. Representative of N = 6 different experiments. B. Effect of vehicle (filled symbol) and cystamine (0.1–1 mM; open symbol) on 5-HT (top) and KCl (bottom)-induced contraction in isolated rat aorta. * indicates statistical difference from vehicle-incubated values. Points and vertical lines represent means±SEM for number of animals in parentheses.

    Techniques Used: Inhibition, Activity Assay, Immunoprecipitation, Isolation, Incubation

    3) Product Images from "The Kampo medicine Yokukansan (YKS) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells"

    Article Title: The Kampo medicine Yokukansan (YKS) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells

    Journal: Bosnian Journal of Basic Medical Sciences

    doi: 10.17305/bjbms.2017.2248

    Effect of YKS on NGF-induced phosphorylation of Akt and ERK1/2 in PC12 cells. (A, C) PC12 cells were treated for various time periods (0, 5, 10, 20, 30, or 60 minutes) with NGF in the presence or absence of 0.5 mg/mL YKS; (A) the whole-cell extracts were subjected to SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane, and treated with primary anti-p-Akt and anti-Akt antibodies, or (C) with primary anti-p-ERK1/2 and anti-ERK1/2 antibodies. The extracts were then treated with horseradish peroxidase-conjugated secondary antibody. (B) The intensity of the p-Akt polypeptide band was normalized to the total Akt band (p-Akt/Akt), whereas (D) the intensity of the p-ERK1/2 polypeptide band was normalized to the total ERK1/2 band (p-ERK1/2/ERK1/2). The results are presented as the mean ± SD of three independent experiments. YKS: Yokukansan; NGF: Nerve growth factor; Akt or PKB: Protein kinase B; ERK1/2: Extracellular-regulated kinase 1/2; SDS: Sodium dodecyl sulfate; PVDF: Polyvinylidene fluoride; p: phospho.
    Figure Legend Snippet: Effect of YKS on NGF-induced phosphorylation of Akt and ERK1/2 in PC12 cells. (A, C) PC12 cells were treated for various time periods (0, 5, 10, 20, 30, or 60 minutes) with NGF in the presence or absence of 0.5 mg/mL YKS; (A) the whole-cell extracts were subjected to SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane, and treated with primary anti-p-Akt and anti-Akt antibodies, or (C) with primary anti-p-ERK1/2 and anti-ERK1/2 antibodies. The extracts were then treated with horseradish peroxidase-conjugated secondary antibody. (B) The intensity of the p-Akt polypeptide band was normalized to the total Akt band (p-Akt/Akt), whereas (D) the intensity of the p-ERK1/2 polypeptide band was normalized to the total ERK1/2 band (p-ERK1/2/ERK1/2). The results are presented as the mean ± SD of three independent experiments. YKS: Yokukansan; NGF: Nerve growth factor; Akt or PKB: Protein kinase B; ERK1/2: Extracellular-regulated kinase 1/2; SDS: Sodium dodecyl sulfate; PVDF: Polyvinylidene fluoride; p: phospho.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    4) Product Images from "Transgenic Expression of Cyclin-Dependent Kinase 4 Results in Epidermal Hyperplasia, Hypertrophy, and Severe Dermal Fibrosis"

    Article Title: Transgenic Expression of Cyclin-Dependent Kinase 4 Results in Epidermal Hyperplasia, Hypertrophy, and Severe Dermal Fibrosis

    Journal: The American Journal of Pathology

    doi:

    Epidermal proliferation in transgenic and normal sibling mice. BrdU incorporation in transgenic ( A ) and normal ( B ) skin was detected in paraffin sections with mouse anti-BrdU antibody and a secondary antibody conjugated with horseradish peroxidase. C: The bars indicate the label index or the percentage of BrdU incorporation in basal cell layer from interfollicular epithelia.
    Figure Legend Snippet: Epidermal proliferation in transgenic and normal sibling mice. BrdU incorporation in transgenic ( A ) and normal ( B ) skin was detected in paraffin sections with mouse anti-BrdU antibody and a secondary antibody conjugated with horseradish peroxidase. C: The bars indicate the label index or the percentage of BrdU incorporation in basal cell layer from interfollicular epithelia.

    Techniques Used: Transgenic Assay, Mouse Assay, BrdU Incorporation Assay

    Keratin expression in transgenic and normal sibling mice. Keratins 1, 5, and 6 were determined on representative paraffin sections of skin from K5CDK4 transgenics ( B , D , and F ) and normal sibling ( A , C , and E ). Specific antibody binding was detected by secondary antibody conjugated with horseradish peroxidase, and the reaction was developed with diaminobenzidine. Keratin 5 ( A , B ), Keratin 1 ( C , D ) and Keratin 6 ( E , F ).
    Figure Legend Snippet: Keratin expression in transgenic and normal sibling mice. Keratins 1, 5, and 6 were determined on representative paraffin sections of skin from K5CDK4 transgenics ( B , D , and F ) and normal sibling ( A , C , and E ). Specific antibody binding was detected by secondary antibody conjugated with horseradish peroxidase, and the reaction was developed with diaminobenzidine. Keratin 5 ( A , B ), Keratin 1 ( C , D ) and Keratin 6 ( E , F ).

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Binding Assay

    Skin phenotype of K5CDK4 transgenic mice. Representative paraffin-sections of skin from K5CDK4 transgenic ( A and C ) and normal sibling ( B and D ) mice were stained with H E. Expression of CDK4 in transgenic ( E ) and wild-type skin ( F ) was determined with specific antibody. Antibody binding was detected by secondary antibody conjugated with horseradish peroxidase, and the reaction was developed with diaminobenzidine. Original magnifications: ×4 ( A and B ), ×40 ( C and D ). The bars indicate the thickness of the dermis ( A and B ). FT, fat tissue.
    Figure Legend Snippet: Skin phenotype of K5CDK4 transgenic mice. Representative paraffin-sections of skin from K5CDK4 transgenic ( A and C ) and normal sibling ( B and D ) mice were stained with H E. Expression of CDK4 in transgenic ( E ) and wild-type skin ( F ) was determined with specific antibody. Antibody binding was detected by secondary antibody conjugated with horseradish peroxidase, and the reaction was developed with diaminobenzidine. Original magnifications: ×4 ( A and B ), ×40 ( C and D ). The bars indicate the thickness of the dermis ( A and B ). FT, fat tissue.

    Techniques Used: Transgenic Assay, Mouse Assay, Staining, Expressing, Binding Assay

    5) Product Images from "Characterization of HMGB1/2 Interactome in Prostate Cancer by Yeast Two Hybrid Approach: Potential Pathobiological Implications"

    Article Title: Characterization of HMGB1/2 Interactome in Prostate Cancer by Yeast Two Hybrid Approach: Potential Pathobiological Implications

    Journal: Cancers

    doi: 10.3390/cancers11111729

    Validation of HMGB1-interactions: ( a ) Cytokeratin-7 co-immunoprecipitation with HMGB1. PC-3 lysates were immunoprecipitated with anti-HMGB1 antibody or normal mouse IgG and immunoblotted with antibodies to Cytokeratin-7 and HMGB1; complete membranes provided as Supplementary Materials Imagen S1 . Protein G horseradish peroxidase (HRP)-labelled was used as a secondary antibody to minimize the signal given by the light and heavy chains of the immunoprecipitation antibody. ( b ) Immunofluorescent localization of HMGB1 in PC-3 cells and comparison to Hoechst-stained nuclei. ( c ) Immunofluorescent co-localization of HMGB1 and Cytokeratin-7 by confocal microscopy in PC-3 cells. HMGB1 is shown in green, and Cytokeratin-7 is in red. Co-localization is seen in yellow by merging. ( d ) Validation of interactions with HNRNPU, SRSF3, and UBC after HMGB1 immunoprecipitation and MS peptide identification.
    Figure Legend Snippet: Validation of HMGB1-interactions: ( a ) Cytokeratin-7 co-immunoprecipitation with HMGB1. PC-3 lysates were immunoprecipitated with anti-HMGB1 antibody or normal mouse IgG and immunoblotted with antibodies to Cytokeratin-7 and HMGB1; complete membranes provided as Supplementary Materials Imagen S1 . Protein G horseradish peroxidase (HRP)-labelled was used as a secondary antibody to minimize the signal given by the light and heavy chains of the immunoprecipitation antibody. ( b ) Immunofluorescent localization of HMGB1 in PC-3 cells and comparison to Hoechst-stained nuclei. ( c ) Immunofluorescent co-localization of HMGB1 and Cytokeratin-7 by confocal microscopy in PC-3 cells. HMGB1 is shown in green, and Cytokeratin-7 is in red. Co-localization is seen in yellow by merging. ( d ) Validation of interactions with HNRNPU, SRSF3, and UBC after HMGB1 immunoprecipitation and MS peptide identification.

    Techniques Used: Immunoprecipitation, Staining, Confocal Microscopy, Mass Spectrometry

    6) Product Images from "Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A"

    Article Title: Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051242

    Suppression of PKD2 up-regulates Tyr307 phosphorylation of PP2A catalytic subunit after TCR stimulation. (A) Human CD4 + T cell clone was stimulated by L cells expressing its cognate ligands in the presence (closed circles) or absence (open circlse) of PKD2 inhibitor Gö6976 (3 µM). (B) Jurkat cells treated with siRNA were stimulated by anti-CD3ε antibody and anti-mouse IgG antibody. Treatment with siRNA2 (closed circles) significantly reduced the PKD2 expression, while siRNA1 (open circles) had almost no effect and hence used as negative controls (right panel). (C) Normal Jurkat cells (open circles) and Jurkat cells expressing GFP-SET-S171A (closed circles) were stimulated by anti-CD3ε antibody and anti-mouse IgG antibody. The latter cells expressed 4.5-fold higher GFP-SET-S171A than the endogenous SET as judged by the intensity of the bands using anti-SET antibody on the Western blot (upper box). Cell lysate were subjected to SDS-PAGE followed by western blot using anti-PP2A phospho-Tyr307 antibody and horseradish peroxidase-conjugated second antibody. The amount of each band was quantified by using ECL and LAS-4000. The same membrane was stripped and reprobed with the anti-PP2A C-subunit antibody to monitor relative abundance of total PP2A protein. Values represent the means ±SD from three independent experiments.
    Figure Legend Snippet: Suppression of PKD2 up-regulates Tyr307 phosphorylation of PP2A catalytic subunit after TCR stimulation. (A) Human CD4 + T cell clone was stimulated by L cells expressing its cognate ligands in the presence (closed circles) or absence (open circlse) of PKD2 inhibitor Gö6976 (3 µM). (B) Jurkat cells treated with siRNA were stimulated by anti-CD3ε antibody and anti-mouse IgG antibody. Treatment with siRNA2 (closed circles) significantly reduced the PKD2 expression, while siRNA1 (open circles) had almost no effect and hence used as negative controls (right panel). (C) Normal Jurkat cells (open circles) and Jurkat cells expressing GFP-SET-S171A (closed circles) were stimulated by anti-CD3ε antibody and anti-mouse IgG antibody. The latter cells expressed 4.5-fold higher GFP-SET-S171A than the endogenous SET as judged by the intensity of the bands using anti-SET antibody on the Western blot (upper box). Cell lysate were subjected to SDS-PAGE followed by western blot using anti-PP2A phospho-Tyr307 antibody and horseradish peroxidase-conjugated second antibody. The amount of each band was quantified by using ECL and LAS-4000. The same membrane was stripped and reprobed with the anti-PP2A C-subunit antibody to monitor relative abundance of total PP2A protein. Values represent the means ±SD from three independent experiments.

    Techniques Used: Expressing, Western Blot, SDS Page

    7) Product Images from "Sulforaphane suppresses PRMT5/MEP50 function in epidermal squamous cell carcinoma leading to reduced tumor formation"

    Article Title: Sulforaphane suppresses PRMT5/MEP50 function in epidermal squamous cell carcinoma leading to reduced tumor formation

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgx044

    MEP50 and PRMT5 are required for SCC-13 proliferation and migration. ( A ) Skin cancer tissue array was stained with anti-MEP50 and binding was visualized using a peroxidase-conjugated secondary antibody. The section shown is from a scalp squamous cell carcinoma. Bar = 10 μm. Similar staining patterns were observed in 50 distinct cancer samples (not shown). ( B ) SCC-13 cells were seeded on coverslips and localization of endogenous MEP50 was visualized. The cells were fixed and co-stained with DAPI (blue) and anti-MEP50 (green). Similar results were observed in each of three experiments. The staining indicates distribution in the nucleus and cytoplasm. ( C ) SCC-13 cells were double electroporated with control-, MEP50- or PRMT5-siRNA, and then 15000 cells were plated per well. After an overnight attachment, cell number was determined (day zero) and at the indicated times thereafter. The values are mean ± SEM ( n = 3). The asterisks indicate a significant difference ( P
    Figure Legend Snippet: MEP50 and PRMT5 are required for SCC-13 proliferation and migration. ( A ) Skin cancer tissue array was stained with anti-MEP50 and binding was visualized using a peroxidase-conjugated secondary antibody. The section shown is from a scalp squamous cell carcinoma. Bar = 10 μm. Similar staining patterns were observed in 50 distinct cancer samples (not shown). ( B ) SCC-13 cells were seeded on coverslips and localization of endogenous MEP50 was visualized. The cells were fixed and co-stained with DAPI (blue) and anti-MEP50 (green). Similar results were observed in each of three experiments. The staining indicates distribution in the nucleus and cytoplasm. ( C ) SCC-13 cells were double electroporated with control-, MEP50- or PRMT5-siRNA, and then 15000 cells were plated per well. After an overnight attachment, cell number was determined (day zero) and at the indicated times thereafter. The values are mean ± SEM ( n = 3). The asterisks indicate a significant difference ( P

    Techniques Used: Migration, Staining, Binding Assay

    8) Product Images from "Cyclin D2 Overexpression in Transgenic Mice Induces Thymic and Epidermal Hyperplasia whereas Cyclin D3 Expression Results Only in Epidermal Hyperplasia"

    Article Title: Cyclin D2 Overexpression in Transgenic Mice Induces Thymic and Epidermal Hyperplasia whereas Cyclin D3 Expression Results Only in Epidermal Hyperplasia

    Journal: The American Journal of Pathology

    doi:

    Skin phenotype of K5D3 transgenic mice. Representative paraffin-sections of skin from K5D3 transgenic mice ( A ) and normal siblings ( B ) were stained with hematoxylin and eosin. Expression of cyclin D3 in transgenic ( C ) and wild-type skin ( D ) was determined with specific antibodies. BrdU incorporation of paraffin sections of transgenic ( E ) and normal ( F ) skin was detected with mouse monoclonal anti-BrdU antibody. Antibody binding was detected by secondary antibody conjugated with horseradish peroxidase, and the reaction was developed with diaminobenzidine. Original magnification, ×200.
    Figure Legend Snippet: Skin phenotype of K5D3 transgenic mice. Representative paraffin-sections of skin from K5D3 transgenic mice ( A ) and normal siblings ( B ) were stained with hematoxylin and eosin. Expression of cyclin D3 in transgenic ( C ) and wild-type skin ( D ) was determined with specific antibodies. BrdU incorporation of paraffin sections of transgenic ( E ) and normal ( F ) skin was detected with mouse monoclonal anti-BrdU antibody. Antibody binding was detected by secondary antibody conjugated with horseradish peroxidase, and the reaction was developed with diaminobenzidine. Original magnification, ×200.

    Techniques Used: Transgenic Assay, Mouse Assay, Staining, Expressing, BrdU Incorporation Assay, Binding Assay

    Skin phenotype of K5D2 transgenic mice. Representative paraffin-sections of skin from K5D2 transgenic mice ( A ) and normal siblings ( B ) were stained with hematoxylin and eosin. Expression of cyclin D2 in transgenic ( C ) and wild-type skin ( D ) was determined with specific antibodies. BrdU incorporation of paraffin sections of transgenic ( E ) and normal ( F ) skin was detected with mouse monoclonal anti-BrdU antibody. Antibody binding was detected by secondary antibody conjugated with horseradish peroxidase, and the reaction was developed with diaminobenzidine. Original magnification, ×200.
    Figure Legend Snippet: Skin phenotype of K5D2 transgenic mice. Representative paraffin-sections of skin from K5D2 transgenic mice ( A ) and normal siblings ( B ) were stained with hematoxylin and eosin. Expression of cyclin D2 in transgenic ( C ) and wild-type skin ( D ) was determined with specific antibodies. BrdU incorporation of paraffin sections of transgenic ( E ) and normal ( F ) skin was detected with mouse monoclonal anti-BrdU antibody. Antibody binding was detected by secondary antibody conjugated with horseradish peroxidase, and the reaction was developed with diaminobenzidine. Original magnification, ×200.

    Techniques Used: Transgenic Assay, Mouse Assay, Staining, Expressing, BrdU Incorporation Assay, Binding Assay

    9) Product Images from "An Important Role for Syndecan-1 in Herpes Simplex Virus Type-1 Induced Cell-to-Cell Fusion and Virus Spread"

    Article Title: An Important Role for Syndecan-1 in Herpes Simplex Virus Type-1 Induced Cell-to-Cell Fusion and Virus Spread

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025252

    Effect of syndecan-1 overexpression, or downreguulation on HSV-1 plaque size. Monolayers of HCE cells were transfected with either control GFP plasmid, full-length wt syndecan-1 (SDC1), or the construct FcR ecto hS1. 24 h post-transfection, cells were infected with HSV-1 (KOS) at an MOI of 0.1 and overlaid with 0.5% methylcellulose medium. (B) 50% confluent HCE cells were transfected with either control scrambled siRNA or syndecan-1 sprecific siRNA. 72 h post-transfection, cells were infected with HSV-1 (KOS) at an MOI of 0.1 and overlaid with 0.5% methylcellulose medium. (A, B). 72 h post-infection cells were fixed and stained using rabbit anti-HSV-1, horseradish peroxidase-conjugated secondary antibody, and 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate. Plaques were measured with a micrometer at the 10× objective (Zeiss Axiovert 200), and the area was calculated by measuring the outline of each plaque using Axioversion software (version 4). Results are expressed as relative plaque size (means ± 1 SD) of two independent experiments conducted in duplicate. Representative plaques from each condition are shown. SDC1 , syndecan-1.
    Figure Legend Snippet: Effect of syndecan-1 overexpression, or downreguulation on HSV-1 plaque size. Monolayers of HCE cells were transfected with either control GFP plasmid, full-length wt syndecan-1 (SDC1), or the construct FcR ecto hS1. 24 h post-transfection, cells were infected with HSV-1 (KOS) at an MOI of 0.1 and overlaid with 0.5% methylcellulose medium. (B) 50% confluent HCE cells were transfected with either control scrambled siRNA or syndecan-1 sprecific siRNA. 72 h post-transfection, cells were infected with HSV-1 (KOS) at an MOI of 0.1 and overlaid with 0.5% methylcellulose medium. (A, B). 72 h post-infection cells were fixed and stained using rabbit anti-HSV-1, horseradish peroxidase-conjugated secondary antibody, and 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate. Plaques were measured with a micrometer at the 10× objective (Zeiss Axiovert 200), and the area was calculated by measuring the outline of each plaque using Axioversion software (version 4). Results are expressed as relative plaque size (means ± 1 SD) of two independent experiments conducted in duplicate. Representative plaques from each condition are shown. SDC1 , syndecan-1.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Construct, Infection, Staining, Software

    Related Articles

    Western Blot:

    Article Title: The Kampo medicine Yokukansan (YKS) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells
    Article Snippet: .. The immunoreactivity was tested using horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and visualized with enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech) and Fuji RX-U X-ray film (Fuji Film, Tokyo, Japan). .. Data were analyzed using ImageJ software.

    Article Title: Characterization of HMGB1/2 Interactome in Prostate Cancer by Yeast Two Hybrid Approach: Potential Pathobiological Implications
    Article Snippet: .. After incubation with the corresponding horseradish peroxidase-conjugated secondary antibody, enhanced chemiluminescence for high sensitivity and long-lasting signal (ECL) Anti-mouse IgG (NXA931 from GE Healthcare Sciences, Chicago, IL, USA) or ECL Anti-rabbit IgG (NA934 from GE Healthcare Sciences, Chicago, IL, USA), protein bands were detected using LuminataTMCrescendo Western HRP Substrate (Millipore Corporation, Burlington, MA, USA) and a ChemiDocTM imager (Bio-Rad laboratories Hercules, CA, USA). .. Immunofluorescence and Confocal Microscopy Cells were plated in 6-well plates, each containing 4 sterile 13-mm glass coverslips.

    Incubation:

    Article Title: Serotonylation of Vascular Proteins Important to Contraction
    Article Snippet: .. Samples were blocked overnight at 4°C in 4% chick egg ovalbulmin [TBS-0.1% Tween+0.025% NaN3, ], washed in TBS-Tween for 20 minutes, and incubated with streptavidin-linked, horseradish peroxidase-conjugated secondary antibody (1∶2000, 1 hr, 4°C GE Healthcare, Piscataway NJ, USA). .. ECL® reagents (GE Healthcare, Piscataway NJ USA) were used to visualize bands.

    Article Title: Characterization of HMGB1/2 Interactome in Prostate Cancer by Yeast Two Hybrid Approach: Potential Pathobiological Implications
    Article Snippet: .. After incubation with the corresponding horseradish peroxidase-conjugated secondary antibody, enhanced chemiluminescence for high sensitivity and long-lasting signal (ECL) Anti-mouse IgG (NXA931 from GE Healthcare Sciences, Chicago, IL, USA) or ECL Anti-rabbit IgG (NA934 from GE Healthcare Sciences, Chicago, IL, USA), protein bands were detected using LuminataTMCrescendo Western HRP Substrate (Millipore Corporation, Burlington, MA, USA) and a ChemiDocTM imager (Bio-Rad laboratories Hercules, CA, USA). .. Immunofluorescence and Confocal Microscopy Cells were plated in 6-well plates, each containing 4 sterile 13-mm glass coverslips.

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    GE Healthcare anti mouse secondary antibody conjugated to hrp
    LGG induces ROS that oxidizes cysteines. (A) Intestinal epithelial cells (SKCO15) were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, and then with 15 µM HydroCy3 for 30 min before confocal microscopic analysis at 555 nm, (scale bars 20 µm). (B) SKCO15 were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, labeled for 30 min with a thiol-reactive, Thiol Tracker™ fluorescence probe, and then analyzed by fluorescence microscopy at 405 nm (scale bars 200 µm). Mean image intensity is shown at bottom left for A and B. (C) Biotinylated-iodoacetamide (BIAM) labeling of cysteine residues in lysates of LGG or E. coli contacted SKCO15 cells, followed by pull-down of labeled residues with <t>streptavidin</t> conjugated agarose and detected by Western blot using <t>HRP</t> conjugated streptavidin as a probe. The relative intensity of each lane of the blot is shown in arbitrary units to the left. Each value was normalized to calnexin that served as a loading control to give the relative oxidation amounts. (D) Dual labeling of LGG or E. coli contacted (15 mins) SKCO15 cultured cells with HydroCy3 (red) and Lysotracker (green). Note co-localization of lysotracker and hydro-Cy3 in LGG contacted cells (bars 10 µm).
    Anti Mouse Secondary Antibody Conjugated To Hrp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare immunoblot analysis
    Punicalagin does not block NLRP3 or caspase-1 activation. ( a ) Intracellular Ca 2+ rise in mouse BMDMs primed with LPS (1 μ g/ml, 4 h) followed by stimulation with ATP (1 mM, added when indicated with an arrow) in the absence or presence of punicalagin (PUN; 25 μ M). ( b ) Relative intracellular K + concentration of BMDMs treated as in ( a ) with ATP for 30 min. ( c ) Kinetic of net BRET signal for NLRP3 protein expressed in P2X7-HEK293 cells unstimulated or stimulated with ATP (5 mM, added when indicated with an arrow). ( d ) Average quantification (top) and fluorescence microscopy images (bottom) of immortalised ASC-Cherry macrophages containing ASC specks treated as in ( b ); n > 400 cells/condition from 2 independent experiments; scale bar represents 20 μ m. ( e and f ) <t>Immunoblot</t> analysis ( e ) and caspase-1 activity measurements ( f ) of cell lysate and supernatant of BMDMs treated as in ( b ); *** P
    Immunoblot Analysis, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare secondary horseradish peroxidase conjugated donkey anti rabbit igg
    The single alanine substitutions of the key amino acid residues within the NR2B and NR1 M3 domains contribute to the regulation of the trafficking of the functional receptors. A, the amino acid sequences of the NR2B and NR1 M3 domains are shown, and the amino acid residues replaced with alanines are underlined. B and C, COS-7 cells cotransfected with indicated NR1/NR2B subunits were solubilized with 1% deoxycholate, immunoprecipitated ( IP ) with mouse anti-NR1 ( B ) or anti-MYC ( C ) antibodies ( Ab ), and probed with rabbit <t>anti-GFP</t> antibody. The specificity of coimmunoprecipitation was tested by using <t>IgG.</t> The images show the representative results from three independent experiments. Densitometric analysis revealed no significant differences in the normalized amounts of the bound fractions among the studied combinations of the subunits (ratio between YFP-NR1-1a-W636A-Y647A-T648A/MYC-NR2B and YFP-NR1-1a/MYC-NR2B, 1.07 ± 0.12; ratio between NR1-1a/GFP-NR2B-W635A-S645A-Y646A-T647A and NR1-1a/GFP-NR2B, 1.09 ± 0.04; n = 3). D and E, the distribution of indicated mutated NMDA receptors closely matches the distribution of an ER marker ( D ) but not a GA marker ( E ). Images were taken on fixed COS-7 cells using a confocal microscope. PDI , oxidoreductase-protein disulfide isomerase.
    Secondary Horseradish Peroxidase Conjugated Donkey Anti Rabbit Igg, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    secondary horseradish peroxidase conjugated donkey anti rabbit igg - by Bioz Stars, 2020-07
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    85
    GE Healthcare horse raddish peroxidase hrp conjugated secondary antibodies sheep anti mouse igg
    Degradation of deposited C3b by B. anthracis spores-bound PLG. (A) Spores (2×10 7 /well) were immobilized onto a microplate and incubated with 10% NHS (100 µl) for 30 min at room temperature. Washed spores were incubated with 2 µg/well PLG for 1 h in the presence or absence of protease cocktail. Bound PLG was activated by uPA (20 units/well) for 3 h at 37°C. Deposited C3b was detected by anti-C3c <t>IgG</t> and <t>HRP-conjugated</t> secondary antibody followed by TMB reaction. C3b deposition is expressed as the mean absorbance at 450 nm of quadruplicates. Error bars indicate ± standard deviation. *P
    Horse Raddish Peroxidase Hrp Conjugated Secondary Antibodies Sheep Anti Mouse Igg, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horse raddish peroxidase hrp conjugated secondary antibodies sheep anti mouse igg/product/GE Healthcare
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    horse raddish peroxidase hrp conjugated secondary antibodies sheep anti mouse igg - by Bioz Stars, 2020-07
    85/100 stars
      Buy from Supplier

    Image Search Results


    LGG induces ROS that oxidizes cysteines. (A) Intestinal epithelial cells (SKCO15) were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, and then with 15 µM HydroCy3 for 30 min before confocal microscopic analysis at 555 nm, (scale bars 20 µm). (B) SKCO15 were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, labeled for 30 min with a thiol-reactive, Thiol Tracker™ fluorescence probe, and then analyzed by fluorescence microscopy at 405 nm (scale bars 200 µm). Mean image intensity is shown at bottom left for A and B. (C) Biotinylated-iodoacetamide (BIAM) labeling of cysteine residues in lysates of LGG or E. coli contacted SKCO15 cells, followed by pull-down of labeled residues with streptavidin conjugated agarose and detected by Western blot using HRP conjugated streptavidin as a probe. The relative intensity of each lane of the blot is shown in arbitrary units to the left. Each value was normalized to calnexin that served as a loading control to give the relative oxidation amounts. (D) Dual labeling of LGG or E. coli contacted (15 mins) SKCO15 cultured cells with HydroCy3 (red) and Lysotracker (green). Note co-localization of lysotracker and hydro-Cy3 in LGG contacted cells (bars 10 µm).

    Journal: Redox Biology

    Article Title: Proteomic analysis of microbial induced redox-dependent intestinal signaling

    doi: 10.1016/j.redox.2018.11.011

    Figure Lengend Snippet: LGG induces ROS that oxidizes cysteines. (A) Intestinal epithelial cells (SKCO15) were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, and then with 15 µM HydroCy3 for 30 min before confocal microscopic analysis at 555 nm, (scale bars 20 µm). (B) SKCO15 were contacted with 10 8 CFU/mL of E. coli or LGG for 15 min, labeled for 30 min with a thiol-reactive, Thiol Tracker™ fluorescence probe, and then analyzed by fluorescence microscopy at 405 nm (scale bars 200 µm). Mean image intensity is shown at bottom left for A and B. (C) Biotinylated-iodoacetamide (BIAM) labeling of cysteine residues in lysates of LGG or E. coli contacted SKCO15 cells, followed by pull-down of labeled residues with streptavidin conjugated agarose and detected by Western blot using HRP conjugated streptavidin as a probe. The relative intensity of each lane of the blot is shown in arbitrary units to the left. Each value was normalized to calnexin that served as a loading control to give the relative oxidation amounts. (D) Dual labeling of LGG or E. coli contacted (15 mins) SKCO15 cultured cells with HydroCy3 (red) and Lysotracker (green). Note co-localization of lysotracker and hydro-Cy3 in LGG contacted cells (bars 10 µm).

    Article Snippet: Anti-mouse secondary antibody conjugated to HRP was obtained from GE, while the streptavidin conjugated HRP was obtained from Abcam.

    Techniques: Labeling, Fluorescence, Microscopy, Western Blot, Cell Culture

    Punicalagin does not block NLRP3 or caspase-1 activation. ( a ) Intracellular Ca 2+ rise in mouse BMDMs primed with LPS (1 μ g/ml, 4 h) followed by stimulation with ATP (1 mM, added when indicated with an arrow) in the absence or presence of punicalagin (PUN; 25 μ M). ( b ) Relative intracellular K + concentration of BMDMs treated as in ( a ) with ATP for 30 min. ( c ) Kinetic of net BRET signal for NLRP3 protein expressed in P2X7-HEK293 cells unstimulated or stimulated with ATP (5 mM, added when indicated with an arrow). ( d ) Average quantification (top) and fluorescence microscopy images (bottom) of immortalised ASC-Cherry macrophages containing ASC specks treated as in ( b ); n > 400 cells/condition from 2 independent experiments; scale bar represents 20 μ m. ( e and f ) Immunoblot analysis ( e ) and caspase-1 activity measurements ( f ) of cell lysate and supernatant of BMDMs treated as in ( b ); *** P

    Journal: Cell Death and Differentiation

    Article Title: Inflammasome-dependent IL-1β release depends upon membrane permeabilisation

    doi: 10.1038/cdd.2015.176

    Figure Lengend Snippet: Punicalagin does not block NLRP3 or caspase-1 activation. ( a ) Intracellular Ca 2+ rise in mouse BMDMs primed with LPS (1 μ g/ml, 4 h) followed by stimulation with ATP (1 mM, added when indicated with an arrow) in the absence or presence of punicalagin (PUN; 25 μ M). ( b ) Relative intracellular K + concentration of BMDMs treated as in ( a ) with ATP for 30 min. ( c ) Kinetic of net BRET signal for NLRP3 protein expressed in P2X7-HEK293 cells unstimulated or stimulated with ATP (5 mM, added when indicated with an arrow). ( d ) Average quantification (top) and fluorescence microscopy images (bottom) of immortalised ASC-Cherry macrophages containing ASC specks treated as in ( b ); n > 400 cells/condition from 2 independent experiments; scale bar represents 20 μ m. ( e and f ) Immunoblot analysis ( e ) and caspase-1 activity measurements ( f ) of cell lysate and supernatant of BMDMs treated as in ( b ); *** P

    Article Snippet: Secondary antibodies for immunoblot analysis (ECL horseradish peroxidase conjugate–linked whole sheep antibody to mouse IgG (NA931V) and ECL horseradish peroxidase conjugate-linked donkey antibody (F(ab′)2 fragment) to rabbit IgG (NA9340V)) were from GE Healthcare (Uppsala, Sweden).

    Techniques: Blocking Assay, Activation Assay, Concentration Assay, Bioluminescence Resonance Energy Transfer, Fluorescence, Microscopy, Activity Assay

    Punicalagin blocks mature IL-1 β release and membrane permeabilisation. ( a ) Immunoblot analysis of the processing and release of pro-IL-1 β in cell lysates and supernatants of LPS-primed (1 μ g/ml, 4 h) BMDM unstimulated (−) or stimulated (+) for 30 min with ATP (5 mM) in the presence (+) or absence (−) of punicalagin (PUN; 25 μ M). ( b ) Kinetics of Yo-Pro uptake in BMDMs treated as in ( a ). ( c ) Punicalagin concentration–inhibition curves obtained for IL-1 β release and Yo-Pro uptake in BMDMs treated as in ( a ); punicalagin IC 50 values were 3.91 and 7.65 μ M for IL-1 β release and Yo-Pro uptake respectively. ( d ) Kinetics of Yo-Pro uptake in LPS-primed BMDMs treated with digitonin (50 μ M) or Triton X-100 (0.1%) in the presence or absence of punicalagin (PUN; 25 μ M). ( e ) Percentage of extracellular LDH from BMDM treated as in ( a ) or for 30 min with detergents as in ( d ) in the presence (+) or absence (−) of punicalagin (PUN; 25 μ M). ** P

    Journal: Cell Death and Differentiation

    Article Title: Inflammasome-dependent IL-1β release depends upon membrane permeabilisation

    doi: 10.1038/cdd.2015.176

    Figure Lengend Snippet: Punicalagin blocks mature IL-1 β release and membrane permeabilisation. ( a ) Immunoblot analysis of the processing and release of pro-IL-1 β in cell lysates and supernatants of LPS-primed (1 μ g/ml, 4 h) BMDM unstimulated (−) or stimulated (+) for 30 min with ATP (5 mM) in the presence (+) or absence (−) of punicalagin (PUN; 25 μ M). ( b ) Kinetics of Yo-Pro uptake in BMDMs treated as in ( a ). ( c ) Punicalagin concentration–inhibition curves obtained for IL-1 β release and Yo-Pro uptake in BMDMs treated as in ( a ); punicalagin IC 50 values were 3.91 and 7.65 μ M for IL-1 β release and Yo-Pro uptake respectively. ( d ) Kinetics of Yo-Pro uptake in LPS-primed BMDMs treated with digitonin (50 μ M) or Triton X-100 (0.1%) in the presence or absence of punicalagin (PUN; 25 μ M). ( e ) Percentage of extracellular LDH from BMDM treated as in ( a ) or for 30 min with detergents as in ( d ) in the presence (+) or absence (−) of punicalagin (PUN; 25 μ M). ** P

    Article Snippet: Secondary antibodies for immunoblot analysis (ECL horseradish peroxidase conjugate–linked whole sheep antibody to mouse IgG (NA931V) and ECL horseradish peroxidase conjugate-linked donkey antibody (F(ab′)2 fragment) to rabbit IgG (NA9340V)) were from GE Healthcare (Uppsala, Sweden).

    Techniques: Concentration Assay, Inhibition

    IL-1 β release pharmacology. ( a ) Immunoblot analysis of cell lysate and supernatant of mouse BMDMs primed with LPS (1 μ g/ml, 4 h), followed by no stimulation (−) or stimulation (+) with ATP (5 mM, 20 min) in the absence (−) or presence (+) of punicalagin (PUN; 25 μ M) and then washout (+) or not (−) for 20 min. ( b ) ELISA of IL-1 β of cell lysate and supernatant from BMDMs primed as in ( a ). Measures are taken every 5 min during 30 min of ATP stimulation (5 mM) after priming (black circles) or washout after 30 min stimulation with ATP (5 mM) with punicalagin (PUN; 25 μ M) (white circles). ( c ) Immunoblot analysis of cell lysate and supernatant of BMDMs treated as in ( a ) and during washout after ATP+PUN cells were incubated with punicalagin (PUN; 25 μ M), in a buffer without Ca 2+ , high K + (150 mM), with NMDG + (0 mM Na + ), or normal ion buffer with BAPTA-AM (100 μ M), E-64 cathepsin inhibitor (50 μ M), Ac-YVAD caspase 1 inhibitor (100 μ M), A438079 P2X7 antagonist (25 μ M), apyrase (3 U/ml), MMP408 (1 μ M), MMP9 (0.5 μ M) and GM6001 (0.5 μ M) metalloprotease inhibitors, TPEN Zn 2+ chelator (50 μ M), colchicine (50 μ M) and cytochalasin B (2.5 μ g/ml), 3-MA autophagy inhibitor (6 mM), U73122 phospholipase C inhibitor (10 μ M), MAFP phospholipase A inhibitor (10 μ M), Ac-DNLD, or Ac-DEVD caspase-3 inhibitors (100 μ M). ( d and e ) Kinetic of Yo-Pro uptake ( d ) and percentage of extracellular LDH ( e ) from macrophages treated as in ( b ). ( f ) Percentage of extracellular LDH release and ELISA of IL-1 β in supernatant from BMDMs treated as in ( a ) and during washout after ATP+PUN cells were incubated with punicalagin (PUN; 25 μ M) or glycine (5 mM). ( g ) Yo-Pro uptake in BMDMs treated as in ( f ). ** P

    Journal: Cell Death and Differentiation

    Article Title: Inflammasome-dependent IL-1β release depends upon membrane permeabilisation

    doi: 10.1038/cdd.2015.176

    Figure Lengend Snippet: IL-1 β release pharmacology. ( a ) Immunoblot analysis of cell lysate and supernatant of mouse BMDMs primed with LPS (1 μ g/ml, 4 h), followed by no stimulation (−) or stimulation (+) with ATP (5 mM, 20 min) in the absence (−) or presence (+) of punicalagin (PUN; 25 μ M) and then washout (+) or not (−) for 20 min. ( b ) ELISA of IL-1 β of cell lysate and supernatant from BMDMs primed as in ( a ). Measures are taken every 5 min during 30 min of ATP stimulation (5 mM) after priming (black circles) or washout after 30 min stimulation with ATP (5 mM) with punicalagin (PUN; 25 μ M) (white circles). ( c ) Immunoblot analysis of cell lysate and supernatant of BMDMs treated as in ( a ) and during washout after ATP+PUN cells were incubated with punicalagin (PUN; 25 μ M), in a buffer without Ca 2+ , high K + (150 mM), with NMDG + (0 mM Na + ), or normal ion buffer with BAPTA-AM (100 μ M), E-64 cathepsin inhibitor (50 μ M), Ac-YVAD caspase 1 inhibitor (100 μ M), A438079 P2X7 antagonist (25 μ M), apyrase (3 U/ml), MMP408 (1 μ M), MMP9 (0.5 μ M) and GM6001 (0.5 μ M) metalloprotease inhibitors, TPEN Zn 2+ chelator (50 μ M), colchicine (50 μ M) and cytochalasin B (2.5 μ g/ml), 3-MA autophagy inhibitor (6 mM), U73122 phospholipase C inhibitor (10 μ M), MAFP phospholipase A inhibitor (10 μ M), Ac-DNLD, or Ac-DEVD caspase-3 inhibitors (100 μ M). ( d and e ) Kinetic of Yo-Pro uptake ( d ) and percentage of extracellular LDH ( e ) from macrophages treated as in ( b ). ( f ) Percentage of extracellular LDH release and ELISA of IL-1 β in supernatant from BMDMs treated as in ( a ) and during washout after ATP+PUN cells were incubated with punicalagin (PUN; 25 μ M) or glycine (5 mM). ( g ) Yo-Pro uptake in BMDMs treated as in ( f ). ** P

    Article Snippet: Secondary antibodies for immunoblot analysis (ECL horseradish peroxidase conjugate–linked whole sheep antibody to mouse IgG (NA931V) and ECL horseradish peroxidase conjugate-linked donkey antibody (F(ab′)2 fragment) to rabbit IgG (NA9340V)) were from GE Healthcare (Uppsala, Sweden).

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation

    The single alanine substitutions of the key amino acid residues within the NR2B and NR1 M3 domains contribute to the regulation of the trafficking of the functional receptors. A, the amino acid sequences of the NR2B and NR1 M3 domains are shown, and the amino acid residues replaced with alanines are underlined. B and C, COS-7 cells cotransfected with indicated NR1/NR2B subunits were solubilized with 1% deoxycholate, immunoprecipitated ( IP ) with mouse anti-NR1 ( B ) or anti-MYC ( C ) antibodies ( Ab ), and probed with rabbit anti-GFP antibody. The specificity of coimmunoprecipitation was tested by using IgG. The images show the representative results from three independent experiments. Densitometric analysis revealed no significant differences in the normalized amounts of the bound fractions among the studied combinations of the subunits (ratio between YFP-NR1-1a-W636A-Y647A-T648A/MYC-NR2B and YFP-NR1-1a/MYC-NR2B, 1.07 ± 0.12; ratio between NR1-1a/GFP-NR2B-W635A-S645A-Y646A-T647A and NR1-1a/GFP-NR2B, 1.09 ± 0.04; n = 3). D and E, the distribution of indicated mutated NMDA receptors closely matches the distribution of an ER marker ( D ) but not a GA marker ( E ). Images were taken on fixed COS-7 cells using a confocal microscope. PDI , oxidoreductase-protein disulfide isomerase.

    Journal: The Journal of Biological Chemistry

    Article Title: Key Amino Acid Residues within the Third Membrane Domains of NR1 and NR2 Subunits Contribute to the Regulation of the Surface Delivery of N-methyl-d-aspartate Receptors *

    doi: 10.1074/jbc.M112.339085

    Figure Lengend Snippet: The single alanine substitutions of the key amino acid residues within the NR2B and NR1 M3 domains contribute to the regulation of the trafficking of the functional receptors. A, the amino acid sequences of the NR2B and NR1 M3 domains are shown, and the amino acid residues replaced with alanines are underlined. B and C, COS-7 cells cotransfected with indicated NR1/NR2B subunits were solubilized with 1% deoxycholate, immunoprecipitated ( IP ) with mouse anti-NR1 ( B ) or anti-MYC ( C ) antibodies ( Ab ), and probed with rabbit anti-GFP antibody. The specificity of coimmunoprecipitation was tested by using IgG. The images show the representative results from three independent experiments. Densitometric analysis revealed no significant differences in the normalized amounts of the bound fractions among the studied combinations of the subunits (ratio between YFP-NR1-1a-W636A-Y647A-T648A/MYC-NR2B and YFP-NR1-1a/MYC-NR2B, 1.07 ± 0.12; ratio between NR1-1a/GFP-NR2B-W635A-S645A-Y646A-T647A and NR1-1a/GFP-NR2B, 1.09 ± 0.04; n = 3). D and E, the distribution of indicated mutated NMDA receptors closely matches the distribution of an ER marker ( D ) but not a GA marker ( E ). Images were taken on fixed COS-7 cells using a confocal microscope. PDI , oxidoreductase-protein disulfide isomerase.

    Article Snippet: Proteins were loaded onto 7% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, incubated with rabbit primary anti-GFP (Millipore, 1:1000) and secondary horseradish peroxidase-conjugated donkey anti-rabbit IgG (GE Healthcare, 1:5000) antibodies, and detected with ECL using BioMax MR x-ray films (Eastman Kodak, Rochester, NY).

    Techniques: Functional Assay, Immunoprecipitation, Marker, Microscopy

    Degradation of deposited C3b by B. anthracis spores-bound PLG. (A) Spores (2×10 7 /well) were immobilized onto a microplate and incubated with 10% NHS (100 µl) for 30 min at room temperature. Washed spores were incubated with 2 µg/well PLG for 1 h in the presence or absence of protease cocktail. Bound PLG was activated by uPA (20 units/well) for 3 h at 37°C. Deposited C3b was detected by anti-C3c IgG and HRP-conjugated secondary antibody followed by TMB reaction. C3b deposition is expressed as the mean absorbance at 450 nm of quadruplicates. Error bars indicate ± standard deviation. *P

    Journal: PLoS ONE

    Article Title: Bacillus anthracis Interacts with Plasmin(ogen) to Evade C3b-Dependent Innate Immunity

    doi: 10.1371/journal.pone.0018119

    Figure Lengend Snippet: Degradation of deposited C3b by B. anthracis spores-bound PLG. (A) Spores (2×10 7 /well) were immobilized onto a microplate and incubated with 10% NHS (100 µl) for 30 min at room temperature. Washed spores were incubated with 2 µg/well PLG for 1 h in the presence or absence of protease cocktail. Bound PLG was activated by uPA (20 units/well) for 3 h at 37°C. Deposited C3b was detected by anti-C3c IgG and HRP-conjugated secondary antibody followed by TMB reaction. C3b deposition is expressed as the mean absorbance at 450 nm of quadruplicates. Error bars indicate ± standard deviation. *P

    Article Snippet: Horse raddish peroxidase (HRP)-conjugated secondary antibodies sheep anti-mouse IgG and donkey anti-rabbit IgG antibody were purchased from GE Healthcare and rabbit anti-goat IgG antibody from Jackson Immuno Research.

    Techniques: Incubation, Standard Deviation