cross adsorbed goat anti mouse igg conjugated to alexa fluor 568  (Thermo Fisher)


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    Name:
    Goat anti Mouse IgG H L cross adsorbed
    Description:
    This product is a sample size 50 µG of Goat anti Mouse IgG H L cross adsorbed horseradish peroxidase HRP Conjugate A16072 Goat Anti Mouse IgG H L Antibody Horseradish Peroxidase HRP Conjugate is prepared from antibodies that have been adsorbed against bovine horse human pig or rabbit serum proteins to minimize cross reactivity Goat Anti Mouse IgG H L Antibody Horseradish Peroxidase HRP Conjugate should be suitable for western blot ELISA immunohistochemistry and other standard immunoassay applications The sensitivity of each lot of antibody is confirmed using ELISA The specificity of each lot of antibody is confirmed by isoelectric focusing IEF Applications Goat Anti Mouse IgG H L Antibody Horseradish Peroxidase HRP Conjugate should be suitable for western blot ELISA immunohistochemistry and other standard immunoassay applications Host species The host species of theanti mouse IgG H L horseradish peroxidase HRP Conjugate is goat Reactivity Goat Anti Mouse IgG H L Antibody Horseradish Peroxidase HRP Conjugate detects mouse IgG H L Product size 50 µg Suggested dilution The optimal working dilution should be determined empirically Dilutions of 1 500 1 5000 should be satisfactory for most immunohistochemical applications Dilutions of 1 200 1 5 000 should be satisfactory for most ELISA and western blot applications
    Catalog Number:
    A16072SAMPLE
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
    Applications:
    Build Your Own Immunoassay|Cell Analysis|Cellular Imaging|ELISA|IHC Staining & Detection|Immunohistochemistry (IHC)|Protein Assays and Analysis|Protein Biology|Ready-To-Use Immunoassay|Western Blot Detection|Western Blotting|Secondary Detection
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    Structured Review

    Thermo Fisher cross adsorbed goat anti mouse igg conjugated to alexa fluor 568
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa <t>Fluor</t> 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    This product is a sample size 50 µG of Goat anti Mouse IgG H L cross adsorbed horseradish peroxidase HRP Conjugate A16072 Goat Anti Mouse IgG H L Antibody Horseradish Peroxidase HRP Conjugate is prepared from antibodies that have been adsorbed against bovine horse human pig or rabbit serum proteins to minimize cross reactivity Goat Anti Mouse IgG H L Antibody Horseradish Peroxidase HRP Conjugate should be suitable for western blot ELISA immunohistochemistry and other standard immunoassay applications The sensitivity of each lot of antibody is confirmed using ELISA The specificity of each lot of antibody is confirmed by isoelectric focusing IEF Applications Goat Anti Mouse IgG H L Antibody Horseradish Peroxidase HRP Conjugate should be suitable for western blot ELISA immunohistochemistry and other standard immunoassay applications Host species The host species of theanti mouse IgG H L horseradish peroxidase HRP Conjugate is goat Reactivity Goat Anti Mouse IgG H L Antibody Horseradish Peroxidase HRP Conjugate detects mouse IgG H L Product size 50 µg Suggested dilution The optimal working dilution should be determined empirically Dilutions of 1 500 1 5000 should be satisfactory for most immunohistochemical applications Dilutions of 1 200 1 5 000 should be satisfactory for most ELISA and western blot applications
    https://www.bioz.com/result/cross adsorbed goat anti mouse igg conjugated to alexa fluor 568/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cross adsorbed goat anti mouse igg conjugated to alexa fluor 568 - by Bioz Stars, 2021-05
    95/100 stars

    Images

    1) Product Images from "Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence"

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00864-13

    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Figure Legend Snippet: Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Techniques Used: Confocal Microscopy, Labeling, Incubation, Infection, Invasion Assay

    Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Figure Legend Snippet: Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Techniques Used: Confocal Microscopy, Clonogenic Cell Survival Assay, Labeling, Infection

    Related Articles

    Incubation:

    Article Title: Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display
    Article Snippet: .. After a 1 hour incubation, the plate was washed and the Nedd8 protein bound to the plate was detected by binding consecutively with a mouse anti-HA antibody (Santa Cruz Biotechnology) (1∶500 dilution in TBS with 3% BSA) and a goat anti-mouse antibody conjugated with HRP (Pierce) (1∶20,000 dilution in TBS with 3% BSA). .. The peroxidase activity immobilized on the plate was assayed by the addition of a 1∶1 mixture of tetramethylbenzidine (TMB) and hydrogen peroxide solution (Thermo Fisher Scientific).

    Article Title: DNAM-1 and PVR Regulate Monocyte Migration through Endothelial Junctions
    Article Snippet: 96-well trays were coated with an antibody against the human Fc fragment (Sigma-Aldrich) at 10 μg/ml. .. After saturation of wells with PBS containing 1% BSA, 10−9 M of PVR-vcc-Fc or PVR-v-Fc was reacted with 2.5 μg/ml of the different mAbs followed by an incubation with a 1/2,000 dilution of a peroxidase-labeled goat anti–mouse and revealed with the One Step ABTS (Pierce Chemical Co.). ..

    Binding Assay:

    Article Title: Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display
    Article Snippet: .. After a 1 hour incubation, the plate was washed and the Nedd8 protein bound to the plate was detected by binding consecutively with a mouse anti-HA antibody (Santa Cruz Biotechnology) (1∶500 dilution in TBS with 3% BSA) and a goat anti-mouse antibody conjugated with HRP (Pierce) (1∶20,000 dilution in TBS with 3% BSA). .. The peroxidase activity immobilized on the plate was assayed by the addition of a 1∶1 mixture of tetramethylbenzidine (TMB) and hydrogen peroxide solution (Thermo Fisher Scientific).

    Staining:

    Article Title: Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer
    Article Snippet: The anti-HA antibody was added to a final concentration of 10 μg mL−1 and the cells were incubated for overnight at 4 °C. .. The cells were then washed twice with 0.1% BSA in TBS and stained with 5 μg mL−1 goat anti-mouse antibody conjugated with Alexa Fluor 647 (Life Technologies, A21235) in 0.1 mL 0.1% BSA in TBS. .. 5 μg mL−1 streptavidin conjugated with PE (Life Technologies, S866) was also added to bind to biotin-UB conjugated to the HECT domain.

    Labeling:

    Article Title: The full amino acid repertoire is superior to serine/tyrosine for selection of high affinity immunoglobulin G binders from the fibronectin scaffold
    Article Snippet: .. The resultant population was labeled with 150 nM mouse anti-c-myc antibody (clone 9E10, Covance, Denver, PA, USA) followed by 25 nM goat anti-mouse phycoerythrin conjugate (Invitrogen). .. Full-length Fn3 clones, represented by cells with a positive phycoerythrin signal, were selected via FACS on an FACS Aria (Becton Dickinson, Franklin Lakes, NJ, USA) or MoFlo (Dako Cytomation, Carpinteria, CA, USA).

    Concentration Assay:

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence
    Article Snippet: This antibody was purified from rabbit serum using HiTrap protein G HP affinity columns (GE Healthcare) and directly conjugated to Alexa Fluor 488 (Life Technologies). .. Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200). .. All antibodies were tested against bacteria alone and H292 cells alone to control for nonspecific staining.

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  • 95
    Thermo Fisher cross adsorbed goat anti mouse igg conjugated to alexa fluor 568
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa <t>Fluor</t> 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Cross Adsorbed Goat Anti Mouse Igg Conjugated To Alexa Fluor 568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cross adsorbed goat anti mouse igg conjugated to alexa fluor 568/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cross adsorbed goat anti mouse igg conjugated to alexa fluor 568 - by Bioz Stars, 2021-05
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    86
    Thermo Fisher goat anti mouse igg1 cross adsorbed secondary antibody
    TonEBP interacts with METTL3 and m6A methylase. ( A ) The TonEBP interactome includes METTL3 and R-loop-related proteins. ( B ) HEK293T cell lysates were immunoprecipitated with normal serum (Serum) or anti-TonEBP antibody (TonEBP). Precipitates and cell lysates were blotted for TonEBP and METTL3. ( C ) Cell lysates were immunoprecipitated with normal rabbit <t>IgG</t> (IgG) or anti-METTL3 IgG (METTL3). ( D ) Domain structures of human TonEBP (WT), and deletion proteins ΔRHD and Yc1. ( E ) Domain structures of human METTL3 (WT) and deletion proteins 1–380, 1–200 and 381–580. ( F ) Cells were transfected with plasmids expressing Flag-METTL3 together with Myc-tagged TonEBP (WT), ΔRHD, or Yc1. After 24 h, cell lysates were prepared and immunoprecipitated using anti-FLAG antibody. ( G ) Cells were transfected with plasmids expressing Myc-Yc1 together with Flag-tagged METTL3 (WT), 1–380, 1–200 or 381–500 and immunoprecipitation was performed with Myc antibody 24 h later. ( H ) Amino-acid sequence alignment of the highly conserved and charged regions of TonEBP from seven species. 3M is a mutant Yc1 in which R, E and R were all exchanged to A shown in red and 5M is another mutant where K, R, and the three Ks were all replaced by A shown in green. ( I ) Cells were transfected with a plasmid expressing Flag-tagged Yc1, 3M or 5M. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody after 24 h incubation.
    Goat Anti Mouse Igg1 Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mouse igg1 cross adsorbed secondary antibody/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
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    97
    Thermo Fisher anti mouse igg antibody conjugated to alexa fluor 568
    Distribution of β-COP and dsRNA in the cells at early times in EMCV, ParV1, and EV11 infections. Cells were infected with EMCV (A to C), ParV1 (D to F), or EV11 (G to I) at an MOI of 3. The infected cells were fixed at 5 h p.i. (EMCV) or 4 h p.i. (ParV1 and EV11), double-labeled with anti-dsRNA and anti-β-COP antibodies, and visualized by confocal IF microscopy. (A, D, and G) Staining with anti-β-COP antibody and <t>Alexa</t> <t>Fluor</t> 568 conjugate (red). (B, E, and H) Staining with anti-dsRNA antibody and Alexa Fluor 488 conjugate (green). (C, F, and I) Merge of first two columns; the sites of colocalization of the two antibodies are highlighted in yellow.
    Anti Mouse Igg Antibody Conjugated To Alexa Fluor 568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse igg antibody conjugated to alexa fluor 568/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
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    98
    Thermo Fisher alexa fluor 568 conjugated goat anti human igg
    LPS treatment increases vascular leakage. A , B Intravenously injected tracer (FITC-albumin, MW = 66 kD) accumulates throughout the entire vestibular system of LPS-treated animals ( B ), but not in controls ( A ). C Capillary leakage of FITC-albumin is significant in LPS-treated mice [ n = 6, P (control vs LPS treatment) = 0.007]. D – G Extravasation of intravenously injected <t>IgG</t> <t>Alexa</t> 568 ( white arrows ) from capillaries, visualized by collagen IV immunostaining ( green ), was observed in LPS-treated mice, but not in controls. The scale bar is 50 μm.
    Alexa Fluor 568 Conjugated Goat Anti Human Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200).

    Techniques: Confocal Microscopy, Labeling, Incubation, Infection, Invasion Assay

    Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200).

    Techniques: Confocal Microscopy, Clonogenic Cell Survival Assay, Labeling, Infection

    TonEBP interacts with METTL3 and m6A methylase. ( A ) The TonEBP interactome includes METTL3 and R-loop-related proteins. ( B ) HEK293T cell lysates were immunoprecipitated with normal serum (Serum) or anti-TonEBP antibody (TonEBP). Precipitates and cell lysates were blotted for TonEBP and METTL3. ( C ) Cell lysates were immunoprecipitated with normal rabbit IgG (IgG) or anti-METTL3 IgG (METTL3). ( D ) Domain structures of human TonEBP (WT), and deletion proteins ΔRHD and Yc1. ( E ) Domain structures of human METTL3 (WT) and deletion proteins 1–380, 1–200 and 381–580. ( F ) Cells were transfected with plasmids expressing Flag-METTL3 together with Myc-tagged TonEBP (WT), ΔRHD, or Yc1. After 24 h, cell lysates were prepared and immunoprecipitated using anti-FLAG antibody. ( G ) Cells were transfected with plasmids expressing Myc-Yc1 together with Flag-tagged METTL3 (WT), 1–380, 1–200 or 381–500 and immunoprecipitation was performed with Myc antibody 24 h later. ( H ) Amino-acid sequence alignment of the highly conserved and charged regions of TonEBP from seven species. 3M is a mutant Yc1 in which R, E and R were all exchanged to A shown in red and 5M is another mutant where K, R, and the three Ks were all replaced by A shown in green. ( I ) Cells were transfected with a plasmid expressing Flag-tagged Yc1, 3M or 5M. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody after 24 h incubation.

    Journal: Nucleic Acids Research

    Article Title: TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution

    doi: 10.1093/nar/gkaa1162

    Figure Lengend Snippet: TonEBP interacts with METTL3 and m6A methylase. ( A ) The TonEBP interactome includes METTL3 and R-loop-related proteins. ( B ) HEK293T cell lysates were immunoprecipitated with normal serum (Serum) or anti-TonEBP antibody (TonEBP). Precipitates and cell lysates were blotted for TonEBP and METTL3. ( C ) Cell lysates were immunoprecipitated with normal rabbit IgG (IgG) or anti-METTL3 IgG (METTL3). ( D ) Domain structures of human TonEBP (WT), and deletion proteins ΔRHD and Yc1. ( E ) Domain structures of human METTL3 (WT) and deletion proteins 1–380, 1–200 and 381–580. ( F ) Cells were transfected with plasmids expressing Flag-METTL3 together with Myc-tagged TonEBP (WT), ΔRHD, or Yc1. After 24 h, cell lysates were prepared and immunoprecipitated using anti-FLAG antibody. ( G ) Cells were transfected with plasmids expressing Myc-Yc1 together with Flag-tagged METTL3 (WT), 1–380, 1–200 or 381–500 and immunoprecipitation was performed with Myc antibody 24 h later. ( H ) Amino-acid sequence alignment of the highly conserved and charged regions of TonEBP from seven species. 3M is a mutant Yc1 in which R, E and R were all exchanged to A shown in red and 5M is another mutant where K, R, and the three Ks were all replaced by A shown in green. ( I ) Cells were transfected with a plasmid expressing Flag-tagged Yc1, 3M or 5M. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody after 24 h incubation.

    Article Snippet: The slides were fixed in 4% paraformaldehyde in 1× PBS and incubated for 1 h at RT with an Alexa Fluor 488–conjugated goat anti-rat IgG antibody (dilution 1:100, A21208; Molecular Probes/Thermo Fisher) or an Alexa Fluor 568–conjugated goat anti-mouse IgG antibody (dilution 1:100, A21124; Molecular Probes/Thermo Fisher).

    Techniques: Immunoprecipitation, Transfection, Expressing, Sequencing, Mutagenesis, Plasmid Preparation, Incubation

    Distribution of β-COP and dsRNA in the cells at early times in EMCV, ParV1, and EV11 infections. Cells were infected with EMCV (A to C), ParV1 (D to F), or EV11 (G to I) at an MOI of 3. The infected cells were fixed at 5 h p.i. (EMCV) or 4 h p.i. (ParV1 and EV11), double-labeled with anti-dsRNA and anti-β-COP antibodies, and visualized by confocal IF microscopy. (A, D, and G) Staining with anti-β-COP antibody and Alexa Fluor 568 conjugate (red). (B, E, and H) Staining with anti-dsRNA antibody and Alexa Fluor 488 conjugate (green). (C, F, and I) Merge of first two columns; the sites of colocalization of the two antibodies are highlighted in yellow.

    Journal: Journal of Virology

    Article Title: Differential Requirements for COPI Coats in Formation of Replication Complexes among Three Genera of Picornaviridae

    doi: 10.1128/JVI.76.21.11113-11122.2002

    Figure Lengend Snippet: Distribution of β-COP and dsRNA in the cells at early times in EMCV, ParV1, and EV11 infections. Cells were infected with EMCV (A to C), ParV1 (D to F), or EV11 (G to I) at an MOI of 3. The infected cells were fixed at 5 h p.i. (EMCV) or 4 h p.i. (ParV1 and EV11), double-labeled with anti-dsRNA and anti-β-COP antibodies, and visualized by confocal IF microscopy. (A, D, and G) Staining with anti-β-COP antibody and Alexa Fluor 568 conjugate (red). (B, E, and H) Staining with anti-dsRNA antibody and Alexa Fluor 488 conjugate (green). (C, F, and I) Merge of first two columns; the sites of colocalization of the two antibodies are highlighted in yellow.

    Article Snippet: Double IF staining was performed using goat anti-guinea pig immunoglobulin G (IgG) antibody conjugated to Alexa Fluor 488 and goat anti-rabbit or anti-mouse IgG antibody conjugated to Alexa Fluor 568 (Molecular Probes).

    Techniques: Infection, Labeling, Microscopy, Staining

    LPS treatment increases vascular leakage. A , B Intravenously injected tracer (FITC-albumin, MW = 66 kD) accumulates throughout the entire vestibular system of LPS-treated animals ( B ), but not in controls ( A ). C Capillary leakage of FITC-albumin is significant in LPS-treated mice [ n = 6, P (control vs LPS treatment) = 0.007]. D – G Extravasation of intravenously injected IgG Alexa 568 ( white arrows ) from capillaries, visualized by collagen IV immunostaining ( green ), was observed in LPS-treated mice, but not in controls. The scale bar is 50 μm.

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    Article Title: Characterization and Inflammatory Response of Perivascular-Resident Macrophage-Like Melanocytes in the Vestibular System

    doi: 10.1007/s10162-013-0403-2

    Figure Lengend Snippet: LPS treatment increases vascular leakage. A , B Intravenously injected tracer (FITC-albumin, MW = 66 kD) accumulates throughout the entire vestibular system of LPS-treated animals ( B ), but not in controls ( A ). C Capillary leakage of FITC-albumin is significant in LPS-treated mice [ n = 6, P (control vs LPS treatment) = 0.007]. D – G Extravasation of intravenously injected IgG Alexa 568 ( white arrows ) from capillaries, visualized by collagen IV immunostaining ( green ), was observed in LPS-treated mice, but not in controls. The scale bar is 50 μm.

    Article Snippet: For in situ detection, Alexa Fluor 568-conjugated goat anti-human IgG (molecular mass, 200 kDa) (A-21090, Invitrogen, USA) was administered by i.v. for 2 h. Anesthetized animals were perfused intravascularly through the left ventricle for 2 min with HBSS followed by 5 min of 4 % paraformaldehyde (PFA) in PBS.

    Techniques: Injection, Mouse Assay, Immunostaining