anti mouse igg antibody conjugated to alexa fluor 568  (Thermo Fisher)


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    Name:
    F ab 2 Goat anti Mouse IgG H L Cross Adsorbed Secondary Antibody
    Description:
    F ab 2 Goat anti Mouse IgG H L Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    Catalog Number:
    31185
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    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.
    F ab 2 Goat anti Mouse IgG H L Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    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
    Price from $9.99 to $1999.99
    anti mouse igg antibody conjugated to alexa fluor 568 - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Differential Requirements for COPI Coats in Formation of Replication Complexes among Three Genera of Picornaviridae"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.21.11113-11122.2002

    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.
    Figure Legend 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.

    Techniques Used: Infection, Labeling, Microscopy, Staining

    Related Articles

    Incubation:

    Article Title: Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes *
    Article Snippet: Cells were subsequently fixed but not permeabilized, and the amount of HA-GLUT4 present at the plasma membrane was determined from the accessibility of the HA epitope to anti-HA antibody (Covance). .. Finally, cells were incubated with 20 μg/ml goat anti-mouse Alexa 488-conjugated secondary antibody (Molecular Probes, Invitrogen). .. After washing, fluorescence (emission 485 nm/excitation 520 nm) was measured in bottom reading mode using a fluorescent microtiter plate reader (FLUOstar Galaxy; BMG Labtechnologies).

    Article Title: Novel Yttria-Stabilized Zirconium Oxide and Lithium Disilicate Coatings on Titanium Alloy Substrate for Implant Abutments and Biomedical Application
    Article Snippet: Soon after, specimens were washed three times for 5 min with a 0.05% Tween-20 solution. .. They were then incubated with secondary goat anti-mouse Alexa Fluor 488-conjugated antibodies (Invitrogen, Carlsbad, CA, USA) and tetramethyl rhodamine iso-thiocyanate (TRITC)-conjugated phalloidin (1:500; Merck Millipore, Carlsbad, CA, USA) in PBS in a darkened environment at RT for 1 h with 25 rpm agitation. .. After that, the samples were washed three more times with PBS for 5 min at RT and stained with 12.5 µg/mL 4’,6-diamidino-2-phenylindole (DAPI; Merck Millipore, Carlsbad, CA, USA) solution in PBS for 5 min in the dark at RT with 25 rpm agitation.

    Article Title: Engagement of Nucleotide-binding Oligomerization Domain-containing Protein 1 (NOD1) by Receptor-interacting Protein 2 (RIP2) Is Insufficient for Signal Transduction *
    Article Snippet: After 24 h, cells were fixed with 3% paraformaldehyde (Roth) in PBS for 10 min and permeabilized with 0.5% Triton X-100 (Roth) in cold PBS for 5 min. .. Cells were blocked in 3% BSA (Roth) in PBS for 20 min and incubated successively in mouse anti-FLAG M2 (1:20,000; Stratagene) and goat anti-mouse Alexa Fluor 546 (1: 200; Invitrogen Molecular Probes) antibodies. .. DNA was stained with DAPI (5 μg/ml; Invitrogen Molecular Probes), and actin was stained with phalloidin-FITC (2.5 μg/ml; Sigma-Aldrich).

    Fluorescence:

    Article Title: Control of Spike Transfer at Hippocampal Mossy Fiber Synapses In Vivo by GABAA and GABAB Receptor-Mediated Inhibition
    Article Snippet: Slices were treated in a blocking buffer [5% normal goat serum in 0.3% PBS–Tween 20 (PBS-T)] for 45 min and incubated with primary antibody solution for 48 h. Slices were then washed and incubated for 2–3 h with a secondary antibody. .. EYFP fluorescence from ChR2+ GCs was enhanced using a mouse anti-GFP (1:500; catalog #11814460, Sigma-Aldrich) and goat anti-mouse conjugated to Alexa Fluor 488 (1:500; catalog # , Thermo Fisher Scientific) diluted in PBS-T. A mounting medium containing DAPI was used (VECTASHIELD, Vector Laboratories). .. The slide scanner was a Nanozoomer 2.0HT with a fluorescence imaging module (Hamamatsu Photonics) using a planar apochromatic 20 × objective with NA of 0.75 combined with an additional lens 1.75×, with a 2 × 2 binning leading to a final magnification of 20×.

    Labeling:

    Article Title: Revealing microbial recognition by specific antibodies
    Article Snippet: Flow cytometry Samples were suspended in sterile saline solution with 5 % albumin to prevent non-specific antibody binding, then stained with (i) anti-human IgA or IgG labelled with FITC (Invitrogen catalog # A18782 and A18806); and (ii) the DNA-binding fluorophor SYTO62 (Invitrogen catalog # S11344) according to the manufacturer instructions. .. Anti-mouse IgA or IgG labeled with FITC (Invitrogen catalog # M31101 and A24525) were used for isotype controls. .. Cell sorting was performed with the MoFloTM XDP flow cytometer (Beckman Coulter Inc.) using Argon 488 nm (blue) laser (200 mW power) and the 635 nm (red) diode laser (25 mW power) as light sources.

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    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
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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
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    86
    Thermo Fisher alexa fluor 568 conjugated goat anti mouse igg
    Induction of autoantibodies against peptides of the second extracellular loop of M3R in mice. Mice were immunized with the M3R-peptides OVA_2ndEL (A, C, E), OVA_2ndEL (B, D, F), or with the OVA peptide control. Antibodies in sera of immunized mice were tested for their binding to immobilized peptides. The titer of total <t>IgG</t> against 2ndEL (A, n = 4 per group) and c2ndEL(B, n = 4 per group) was determined in serum (the dilution factor as depicted) of mice 20weeks after immunization. The subclass of bound IgG, including IgG1, IgG2a, IgG2b and IgG3 against 2ndEL (C) and c2ndEL(D) was determined in samples analyzed in parallel. E and F represent time kinetics of autoantibody production against different peptides of M3R including N terminal (N ter ), the first extracellular loop (1stEL) and the second extracellular loop (2ndEL or c2ndEL) in E) mice immunized with OVA_2ndEL peptide (n = 23)and controls (n = 15) or F) mice immunized with OVA_c2ndEL peptide (n = 8) and control (8). (G) Representative picture showing the IgG-deposition on salivary gland tissue of mice immunized with OVA-2ndEL, OVA-c2ndEL or OVA peptides. The IgG binding was detected by using direct immunofluorescence using DyLightTM649 Goat-anti-Mouse IgG antibody. (H) Representative FACS histogram showing the binding of IgG from OVA, OVA_2ndEL or OVA_c2ndEL immunized mice onto the primary murine salivary gland cells. Primary murine salivary gland cells were incubated with 1:200 diluted sera from OVA (n = 8), OVA_2ndEL (n = 8) or OVA_c2ndEL (n = 8). The bound IgG were detected by <t>Alexa</t> <t>Fluor</t> 568 conjugated goat-anti-mouse IgG. (I and J) Quantified IgG bound onto primary murine gland cells. All data are presented as mean±SD. P values were calculated using Student's t test.
    Alexa Fluor 568 Conjugated Goat Anti Mouse Igg, 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
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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

    Induction of autoantibodies against peptides of the second extracellular loop of M3R in mice. Mice were immunized with the M3R-peptides OVA_2ndEL (A, C, E), OVA_2ndEL (B, D, F), or with the OVA peptide control. Antibodies in sera of immunized mice were tested for their binding to immobilized peptides. The titer of total IgG against 2ndEL (A, n = 4 per group) and c2ndEL(B, n = 4 per group) was determined in serum (the dilution factor as depicted) of mice 20weeks after immunization. The subclass of bound IgG, including IgG1, IgG2a, IgG2b and IgG3 against 2ndEL (C) and c2ndEL(D) was determined in samples analyzed in parallel. E and F represent time kinetics of autoantibody production against different peptides of M3R including N terminal (N ter ), the first extracellular loop (1stEL) and the second extracellular loop (2ndEL or c2ndEL) in E) mice immunized with OVA_2ndEL peptide (n = 23)and controls (n = 15) or F) mice immunized with OVA_c2ndEL peptide (n = 8) and control (8). (G) Representative picture showing the IgG-deposition on salivary gland tissue of mice immunized with OVA-2ndEL, OVA-c2ndEL or OVA peptides. The IgG binding was detected by using direct immunofluorescence using DyLightTM649 Goat-anti-Mouse IgG antibody. (H) Representative FACS histogram showing the binding of IgG from OVA, OVA_2ndEL or OVA_c2ndEL immunized mice onto the primary murine salivary gland cells. Primary murine salivary gland cells were incubated with 1:200 diluted sera from OVA (n = 8), OVA_2ndEL (n = 8) or OVA_c2ndEL (n = 8). The bound IgG were detected by Alexa Fluor 568 conjugated goat-anti-mouse IgG. (I and J) Quantified IgG bound onto primary murine gland cells. All data are presented as mean±SD. P values were calculated using Student's t test.

    Journal: PLoS ONE

    Article Title: Autoantibodies against the Second Extracellular Loop of M3R Do neither Induce nor Indicate Primary Sjögren’s Syndrome

    doi: 10.1371/journal.pone.0149485

    Figure Lengend Snippet: Induction of autoantibodies against peptides of the second extracellular loop of M3R in mice. Mice were immunized with the M3R-peptides OVA_2ndEL (A, C, E), OVA_2ndEL (B, D, F), or with the OVA peptide control. Antibodies in sera of immunized mice were tested for their binding to immobilized peptides. The titer of total IgG against 2ndEL (A, n = 4 per group) and c2ndEL(B, n = 4 per group) was determined in serum (the dilution factor as depicted) of mice 20weeks after immunization. The subclass of bound IgG, including IgG1, IgG2a, IgG2b and IgG3 against 2ndEL (C) and c2ndEL(D) was determined in samples analyzed in parallel. E and F represent time kinetics of autoantibody production against different peptides of M3R including N terminal (N ter ), the first extracellular loop (1stEL) and the second extracellular loop (2ndEL or c2ndEL) in E) mice immunized with OVA_2ndEL peptide (n = 23)and controls (n = 15) or F) mice immunized with OVA_c2ndEL peptide (n = 8) and control (8). (G) Representative picture showing the IgG-deposition on salivary gland tissue of mice immunized with OVA-2ndEL, OVA-c2ndEL or OVA peptides. The IgG binding was detected by using direct immunofluorescence using DyLightTM649 Goat-anti-Mouse IgG antibody. (H) Representative FACS histogram showing the binding of IgG from OVA, OVA_2ndEL or OVA_c2ndEL immunized mice onto the primary murine salivary gland cells. Primary murine salivary gland cells were incubated with 1:200 diluted sera from OVA (n = 8), OVA_2ndEL (n = 8) or OVA_c2ndEL (n = 8). The bound IgG were detected by Alexa Fluor 568 conjugated goat-anti-mouse IgG. (I and J) Quantified IgG bound onto primary murine gland cells. All data are presented as mean±SD. P values were calculated using Student's t test.

    Article Snippet: After washing, cells were incubated in 100 μl staining buffer containing 0.1 μg Alexa Fluor 568 conjugated goat-anti-mouse IgG (Life technologies, USA) for 30 min at 4 degrees and finally suspended in 500 ul staining buffer containing 1% PFA.

    Techniques: Mouse Assay, Binding Assay, Immunofluorescence, FACS, Incubation