non-denaturing polyacrylamide gel electrophoresis Search Results


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  • 97
    Thermo Fisher native page sample buffer
    Reduced E-cadherin–mediated cell adhesion by a constitutively monomeric E-cadherin/αE-catenin chimera. (A) Schematic representation of E-cadΔ70/β/α, in which residues 118–151 of β-catenin are inserted in between E-cadherin and αE-catenin. (B) SEC of E-cadΔ70/β/α (black line), E-cadΔ70/α dimer (green dotted line), and monomer (purple dotted line). (C) <t>CBB-stained</t> <t>Native-PAGE</t> of increasing concentrations of E-cadΔ70/β/α that were incubated for 16 h at 37°C. Quantification of the percent dimerization of E-cadΔ70/β/α compared with E-cadΔ70/α. n = 3. (D) Coimmunoprecipitation of Myc tagged with HA-tagged chimeras, using either E-cadΔ70/α or E-cadΔ70/β/α. Upon immunoprecipitation with HA antibodies, proteins were separated by SDS-PAGE and immunoblotted for HA and Myc. Quantification of the relative binding between differentially tagged E-cadΔ70/α and E-cadΔ70/β/α is shown. n = 4. (E) Immunofluorescence of E-cadΔ70/α and E-cadΔ70/β/α in L cells with an antibody to the extracellular domain of E-cadherin. (F) Adhesion to E-cadherin-Fc of L cells transfected with E-cadherin-GFP, E-cadΔ70/α, E-cadΔ70/β/α, or empty vector control, together with luciferase. Background adhesion to a no calcium control was subtracted, and the mean percentage of adherent cells relative to the total input luciferase signal and normalized to E-cadherin-GFP values is shown. n = 5 with standard deviation. An unpaired Student’s t test was performed for statistical analysis. (G) Hanging drop assay of L cells transfected with E-cadherin-GFP, E-cadΔ70/α, E-cadΔ70/β/α, or empty vector control showing the percentage of clusters of four or more cells, normalized to levels observed with E-cadherin. n = 3, with standard deviation. An unpaired Student’s t test was performed for statistical analysis.
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    Millipore native polyacrylamide gel electrophoresis
    Reduced E-cadherin–mediated cell adhesion by a constitutively monomeric E-cadherin/αE-catenin chimera. (A) Schematic representation of E-cadΔ70/β/α, in which residues 118–151 of β-catenin are inserted in between E-cadherin and αE-catenin. (B) SEC of E-cadΔ70/β/α (black line), E-cadΔ70/α dimer (green dotted line), and monomer (purple dotted line). (C) <t>CBB-stained</t> <t>Native-PAGE</t> of increasing concentrations of E-cadΔ70/β/α that were incubated for 16 h at 37°C. Quantification of the percent dimerization of E-cadΔ70/β/α compared with E-cadΔ70/α. n = 3. (D) Coimmunoprecipitation of Myc tagged with HA-tagged chimeras, using either E-cadΔ70/α or E-cadΔ70/β/α. Upon immunoprecipitation with HA antibodies, proteins were separated by SDS-PAGE and immunoblotted for HA and Myc. Quantification of the relative binding between differentially tagged E-cadΔ70/α and E-cadΔ70/β/α is shown. n = 4. (E) Immunofluorescence of E-cadΔ70/α and E-cadΔ70/β/α in L cells with an antibody to the extracellular domain of E-cadherin. (F) Adhesion to E-cadherin-Fc of L cells transfected with E-cadherin-GFP, E-cadΔ70/α, E-cadΔ70/β/α, or empty vector control, together with luciferase. Background adhesion to a no calcium control was subtracted, and the mean percentage of adherent cells relative to the total input luciferase signal and normalized to E-cadherin-GFP values is shown. n = 5 with standard deviation. An unpaired Student’s t test was performed for statistical analysis. (G) Hanging drop assay of L cells transfected with E-cadherin-GFP, E-cadΔ70/α, E-cadΔ70/β/α, or empty vector control showing the percentage of clusters of four or more cells, normalized to levels observed with E-cadherin. n = 3, with standard deviation. An unpaired Student’s t test was performed for statistical analysis.
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    Bio-Rad non denaturing polyacrylamide gel electrophoresis native page
    ( I ) <t>SDS-PAGE</t> analysis of the <t>PPO</t> protein: M: Marker, 1A: native PPO, 2A: 25 °C-treated protein (thermal treatment), 3A: 25 °C-treated protein (HPCD treatment), 4A: 65 °C-treated protein (thermal treatment), 5A: 65 °C-treated protein (HPCD treatment); ( II ) Native PAGE analysis of PPO protein: M: Marker, 1B: native PPO, 2B: 25 °C-treated protein (thermal treatment). 3B: 25 °C-treated protein (HPCD treatment), 4B: 65 °C-treated protein (thermal treatment), 5B: 65 °C-treated protein (HPCD treatment).
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    91
    Thermo Fisher non denaturing page
    Native <t>PAGE</t> of <t>apolipoprotein-LPS</t> incubations. Apolipoproteins (20 μg) were incubated with increasing amounts of LPS for 1 h and analyzed by PAGE under non-denaturing conditions. Shown are apoA-I (lane 1), incubated with 80 μg (lane 2), 130 μg (lane 3) and 180 μg LPS (lane 4). Lanes 5–8 contain chimera only (lane 5) with 80 μg (lane 6), 130 μg (lane 7) and 180 μg LPS (lane 8); lanes 9–12 contain apoLp-III (lane 9) with 80 μg (lane 10), 130 μg (lane 11) and 180 μg LPS (lane 12).
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    GE Healthcare non denaturing page
    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in <t>SDS-PAGE</t> with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
    Non Denaturing Page, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad native page sample buffer
    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in <t>SDS-PAGE</t> with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
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    Thermo Fisher native page novex
    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in <t>SDS-PAGE</t> with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
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    Thermo Fisher native page buffer
    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in <t>SDS-PAGE</t> with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
    Native Page Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bio-Rad non denaturing tbe gel electrophoresis
    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in <t>SDS-PAGE</t> with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
    Non Denaturing Tbe Gel Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore native page kit
    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in <t>SDS-PAGE</t> with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
    Native Page Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher native page bistris gel
    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in <t>SDS-PAGE</t> with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
    Native Page Bistris Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher native page novex gel
    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in <t>SDS-PAGE</t> with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
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    Bio-Rad native page ready gel
    The <t>pIgR</t> associated IgM–ovalbumin (OVA) complexes in intestinal mucus of group 1 at 6 h after OVA challenge. (A,B) The <t>Native-PAGE</t> under non-reducing condition that stained by Coomassie blue and analyzed by western blot. (A) Lane 1: maker, lanes 2–7: native-PAGE result of serum and gut mucus of group 1 (2–3), group 2 (4–5), and group 3 (6–7), respectively. (B) Western blot result. Lane 1: maker, lanes 2–7: the samples of (A) reacted with anti-IgM monoclonal antibody (MAb) 2D8; lanes 8–13: the samples of (A) reacted with rabbit anti-OVA antibody; lanes 14–19: the samples of (A) reacted with mouse anti-pIgR antibody. (C) SDS-PAGE under reducing condition analyzed the samples from co-immunoprecipitation of gut mucus with rabbit anti-OVA antibody after challenge. Lane 1: maker; lane 2: protein G + rabbit anti-OVA antibody + gut mucus of group 1; lane 3: protein G + rabbit anti-OVA antibody + gut mucus of group 2; lane 4: protein G + rabbit anti-OVA antibody + gut mucus of group 3; lane 5: protein G + non-immune rabbit serum + gut mucus of group 1; lane 6: protein G + gut mucus of group 1. (D) Co-immunoprecipitation of gut mucus with anti-OVA antibody, followed by western blotting assay under reducing conditions. Lane 1: maker; lanes 2–6: Samples of (C) reacted with mouse anti-pIgR antibody; lanes 7–11: Samples of (C) reacted with anti-IgM MAb 2D8.
    Native Page Ready Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad native page mini protean tgx
    The <t>pIgR</t> associated IgM–ovalbumin (OVA) complexes in intestinal mucus of group 1 at 6 h after OVA challenge. (A,B) The <t>Native-PAGE</t> under non-reducing condition that stained by Coomassie blue and analyzed by western blot. (A) Lane 1: maker, lanes 2–7: native-PAGE result of serum and gut mucus of group 1 (2–3), group 2 (4–5), and group 3 (6–7), respectively. (B) Western blot result. Lane 1: maker, lanes 2–7: the samples of (A) reacted with anti-IgM monoclonal antibody (MAb) 2D8; lanes 8–13: the samples of (A) reacted with rabbit anti-OVA antibody; lanes 14–19: the samples of (A) reacted with mouse anti-pIgR antibody. (C) SDS-PAGE under reducing condition analyzed the samples from co-immunoprecipitation of gut mucus with rabbit anti-OVA antibody after challenge. Lane 1: maker; lane 2: protein G + rabbit anti-OVA antibody + gut mucus of group 1; lane 3: protein G + rabbit anti-OVA antibody + gut mucus of group 2; lane 4: protein G + rabbit anti-OVA antibody + gut mucus of group 3; lane 5: protein G + non-immune rabbit serum + gut mucus of group 1; lane 6: protein G + gut mucus of group 1. (D) Co-immunoprecipitation of gut mucus with anti-OVA antibody, followed by western blotting assay under reducing conditions. Lane 1: maker; lanes 2–6: Samples of (C) reacted with mouse anti-pIgR antibody; lanes 7–11: Samples of (C) reacted with anti-IgM MAb 2D8.
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    Image Search Results


    Reduced E-cadherin–mediated cell adhesion by a constitutively monomeric E-cadherin/αE-catenin chimera. (A) Schematic representation of E-cadΔ70/β/α, in which residues 118–151 of β-catenin are inserted in between E-cadherin and αE-catenin. (B) SEC of E-cadΔ70/β/α (black line), E-cadΔ70/α dimer (green dotted line), and monomer (purple dotted line). (C) CBB-stained Native-PAGE of increasing concentrations of E-cadΔ70/β/α that were incubated for 16 h at 37°C. Quantification of the percent dimerization of E-cadΔ70/β/α compared with E-cadΔ70/α. n = 3. (D) Coimmunoprecipitation of Myc tagged with HA-tagged chimeras, using either E-cadΔ70/α or E-cadΔ70/β/α. Upon immunoprecipitation with HA antibodies, proteins were separated by SDS-PAGE and immunoblotted for HA and Myc. Quantification of the relative binding between differentially tagged E-cadΔ70/α and E-cadΔ70/β/α is shown. n = 4. (E) Immunofluorescence of E-cadΔ70/α and E-cadΔ70/β/α in L cells with an antibody to the extracellular domain of E-cadherin. (F) Adhesion to E-cadherin-Fc of L cells transfected with E-cadherin-GFP, E-cadΔ70/α, E-cadΔ70/β/α, or empty vector control, together with luciferase. Background adhesion to a no calcium control was subtracted, and the mean percentage of adherent cells relative to the total input luciferase signal and normalized to E-cadherin-GFP values is shown. n = 5 with standard deviation. An unpaired Student’s t test was performed for statistical analysis. (G) Hanging drop assay of L cells transfected with E-cadherin-GFP, E-cadΔ70/α, E-cadΔ70/β/α, or empty vector control showing the percentage of clusters of four or more cells, normalized to levels observed with E-cadherin. n = 3, with standard deviation. An unpaired Student’s t test was performed for statistical analysis.

    Journal: The Journal of Cell Biology

    Article Title: Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras

    doi: 10.1083/jcb.201411080

    Figure Lengend Snippet: Reduced E-cadherin–mediated cell adhesion by a constitutively monomeric E-cadherin/αE-catenin chimera. (A) Schematic representation of E-cadΔ70/β/α, in which residues 118–151 of β-catenin are inserted in between E-cadherin and αE-catenin. (B) SEC of E-cadΔ70/β/α (black line), E-cadΔ70/α dimer (green dotted line), and monomer (purple dotted line). (C) CBB-stained Native-PAGE of increasing concentrations of E-cadΔ70/β/α that were incubated for 16 h at 37°C. Quantification of the percent dimerization of E-cadΔ70/β/α compared with E-cadΔ70/α. n = 3. (D) Coimmunoprecipitation of Myc tagged with HA-tagged chimeras, using either E-cadΔ70/α or E-cadΔ70/β/α. Upon immunoprecipitation with HA antibodies, proteins were separated by SDS-PAGE and immunoblotted for HA and Myc. Quantification of the relative binding between differentially tagged E-cadΔ70/α and E-cadΔ70/β/α is shown. n = 4. (E) Immunofluorescence of E-cadΔ70/α and E-cadΔ70/β/α in L cells with an antibody to the extracellular domain of E-cadherin. (F) Adhesion to E-cadherin-Fc of L cells transfected with E-cadherin-GFP, E-cadΔ70/α, E-cadΔ70/β/α, or empty vector control, together with luciferase. Background adhesion to a no calcium control was subtracted, and the mean percentage of adherent cells relative to the total input luciferase signal and normalized to E-cadherin-GFP values is shown. n = 5 with standard deviation. An unpaired Student’s t test was performed for statistical analysis. (G) Hanging drop assay of L cells transfected with E-cadherin-GFP, E-cadΔ70/α, E-cadΔ70/β/α, or empty vector control showing the percentage of clusters of four or more cells, normalized to levels observed with E-cadherin. n = 3, with standard deviation. An unpaired Student’s t test was performed for statistical analysis.

    Article Snippet: Before Native-PAGE, samples were diluted to a total concentration of either 1.5 µM (for CBB staining) or 150 nM (for Western blotting) in ice-cold Native-PAGE Sample buffer (Thermo Fisher Scientific).

    Techniques: Size-exclusion Chromatography, Staining, Clear Native PAGE, Incubation, Immunoprecipitation, SDS Page, Binding Assay, Immunofluorescence, Transfection, Plasmid Preparation, Luciferase, Standard Deviation

    E-cadΔ70/α homodimerization is required for robust interaction with F-actin. (A) Schematic representation of the E-cadherin/αE-catenin chimeras. CBD, β-catenin-binding domain. (B) Ion exchange chromatography (IEC) of recombinant E-cadΔ70/α, and SDS-PAGE of protein from the resulting two peaks (fractions indicated in purple and green) stained with Coomassie Brilliant Blue (CBB). (C) Superdex 200 size exclusion chromatography of the two peaks from the IEC shown in B. Fractions indicated with a bracket were pooled and analyzed by Native-PAGE, and stained with CBB. (D) CBB stained Native-PAGE of increasing concentrations of monomeric E-cadΔ70/α chimera incubated for 16 h at 37°C. Ctrl, purified monomeric chimera. Quantification of the percentage of dimerization with standard deviation from three independent experiments. (E) Coimmunoprecipitation of Myc-tagged E-cadΔ70/α with HA-tagged E-cadΔ70/α from transfected L cells. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted for HA and Myc. A representative image of three independent experiments is shown. (F) High-speed co-sedimentation of F-actin with E-cadΔ70/α monomer (purple) or homodimer (green). The data shown are from a single representative experiment out of three independent experiments. (G) Pyrene–actin polymerization assay with 10% pyrene–actin (white), with Arp2/3 complex and WASp-VCA (black), and either 8 µM E-cadΔ70/α homodimer (green) or monomer (purple). The data shown are from a single representative experiment out of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras

    doi: 10.1083/jcb.201411080

    Figure Lengend Snippet: E-cadΔ70/α homodimerization is required for robust interaction with F-actin. (A) Schematic representation of the E-cadherin/αE-catenin chimeras. CBD, β-catenin-binding domain. (B) Ion exchange chromatography (IEC) of recombinant E-cadΔ70/α, and SDS-PAGE of protein from the resulting two peaks (fractions indicated in purple and green) stained with Coomassie Brilliant Blue (CBB). (C) Superdex 200 size exclusion chromatography of the two peaks from the IEC shown in B. Fractions indicated with a bracket were pooled and analyzed by Native-PAGE, and stained with CBB. (D) CBB stained Native-PAGE of increasing concentrations of monomeric E-cadΔ70/α chimera incubated for 16 h at 37°C. Ctrl, purified monomeric chimera. Quantification of the percentage of dimerization with standard deviation from three independent experiments. (E) Coimmunoprecipitation of Myc-tagged E-cadΔ70/α with HA-tagged E-cadΔ70/α from transfected L cells. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted for HA and Myc. A representative image of three independent experiments is shown. (F) High-speed co-sedimentation of F-actin with E-cadΔ70/α monomer (purple) or homodimer (green). The data shown are from a single representative experiment out of three independent experiments. (G) Pyrene–actin polymerization assay with 10% pyrene–actin (white), with Arp2/3 complex and WASp-VCA (black), and either 8 µM E-cadΔ70/α homodimer (green) or monomer (purple). The data shown are from a single representative experiment out of three independent experiments.

    Article Snippet: Before Native-PAGE, samples were diluted to a total concentration of either 1.5 µM (for CBB staining) or 150 nM (for Western blotting) in ice-cold Native-PAGE Sample buffer (Thermo Fisher Scientific).

    Techniques: Binding Assay, Ion Exchange Chromatography, Recombinant, SDS Page, Staining, Size-exclusion Chromatography, Clear Native PAGE, Incubation, Purification, Standard Deviation, Transfection, Immunoprecipitation, Sedimentation, Polymerization Assay

    Complex formation between E-cadherin/αE-catenin chimeras and β-catenin. Superdex 200 gel filtration chromatography of complex formation between β-catenin and E-cadΔ70/α monomer (A), E-cadΔ70/α homodimer (B), E-cad/α monomer (E), or E-cad/α homodimer (F). Proteins were incubated at 25°C either by themselves (colored lines) or together in a 1:1 molar ratio (black lines), and fractions from the individual S200 runs were analyzed by SDS-PAGE and CBB staining. A schematic representation of the complex formed between each chimera and β-catenin is shown. Native-PAGE of monomers and homodimers of E-cadΔ70/α (C) or E-cad/α (G) incubated with β-catenin and immunoblotted for αE-catenin (green) and β-catenin (red). Complex formation is indicated by a shift in band migration and co-fluorescence with both αE-catenin and β-catenin antibodies (heterodimer, solid box; tetramer, dashed box). Note that all proteins in C were run on the same Native-PAGE gel, but the brightness for the last lane was adjusted independently, as indicated. The gel images shown (C and G) are representative of four independent experiments. (D) High-speed co-sedimentation assay of E-cadΔ70/α-β-catenin heterodimer (black triangle) with F-actin. The data shown are from a single representative experiment out of three independent experiments. E-cadΔ70/α dimer (green triangle) and E-cadΔ70/α monomer (purple triangle) from F are shown for comparison.

    Journal: The Journal of Cell Biology

    Article Title: Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras

    doi: 10.1083/jcb.201411080

    Figure Lengend Snippet: Complex formation between E-cadherin/αE-catenin chimeras and β-catenin. Superdex 200 gel filtration chromatography of complex formation between β-catenin and E-cadΔ70/α monomer (A), E-cadΔ70/α homodimer (B), E-cad/α monomer (E), or E-cad/α homodimer (F). Proteins were incubated at 25°C either by themselves (colored lines) or together in a 1:1 molar ratio (black lines), and fractions from the individual S200 runs were analyzed by SDS-PAGE and CBB staining. A schematic representation of the complex formed between each chimera and β-catenin is shown. Native-PAGE of monomers and homodimers of E-cadΔ70/α (C) or E-cad/α (G) incubated with β-catenin and immunoblotted for αE-catenin (green) and β-catenin (red). Complex formation is indicated by a shift in band migration and co-fluorescence with both αE-catenin and β-catenin antibodies (heterodimer, solid box; tetramer, dashed box). Note that all proteins in C were run on the same Native-PAGE gel, but the brightness for the last lane was adjusted independently, as indicated. The gel images shown (C and G) are representative of four independent experiments. (D) High-speed co-sedimentation assay of E-cadΔ70/α-β-catenin heterodimer (black triangle) with F-actin. The data shown are from a single representative experiment out of three independent experiments. E-cadΔ70/α dimer (green triangle) and E-cadΔ70/α monomer (purple triangle) from F are shown for comparison.

    Article Snippet: Before Native-PAGE, samples were diluted to a total concentration of either 1.5 µM (for CBB staining) or 150 nM (for Western blotting) in ice-cold Native-PAGE Sample buffer (Thermo Fisher Scientific).

    Techniques: Filtration, Chromatography, Incubation, SDS Page, Staining, Clear Native PAGE, Migration, Fluorescence, Sedimentation

    ( I ) SDS-PAGE analysis of the PPO protein: M: Marker, 1A: native PPO, 2A: 25 °C-treated protein (thermal treatment), 3A: 25 °C-treated protein (HPCD treatment), 4A: 65 °C-treated protein (thermal treatment), 5A: 65 °C-treated protein (HPCD treatment); ( II ) Native PAGE analysis of PPO protein: M: Marker, 1B: native PPO, 2B: 25 °C-treated protein (thermal treatment). 3B: 25 °C-treated protein (HPCD treatment), 4B: 65 °C-treated protein (thermal treatment), 5B: 65 °C-treated protein (HPCD treatment).

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Inactivation, Aggregation and Conformational Changes of Polyphenol Oxidase from Quince (Cydonia oblonga Miller) Juice Subjected to Thermal and High-Pressure Carbon Dioxide Treatment

    doi: 10.3390/molecules23071743

    Figure Lengend Snippet: ( I ) SDS-PAGE analysis of the PPO protein: M: Marker, 1A: native PPO, 2A: 25 °C-treated protein (thermal treatment), 3A: 25 °C-treated protein (HPCD treatment), 4A: 65 °C-treated protein (thermal treatment), 5A: 65 °C-treated protein (HPCD treatment); ( II ) Native PAGE analysis of PPO protein: M: Marker, 1B: native PPO, 2B: 25 °C-treated protein (thermal treatment). 3B: 25 °C-treated protein (HPCD treatment), 4B: 65 °C-treated protein (thermal treatment), 5B: 65 °C-treated protein (HPCD treatment).

    Article Snippet: Electrophoretic Analysis of Thermal and HPCD-Treated PPO Enzymes Sodium dodecyl sulphate (SDS) and non-denaturing polyacrylamide gel electrophoresis (Native-PAGE) was used to identified the purification process by using the Mini-proten 4 cell system (Bio-Rad, Hercules, CA, USA) to determine the molecular mass of purified PPO [ ].

    Techniques: SDS Page, Marker, Clear Native PAGE

    Trimer stability of the PCNA mutant proteins. ( A ) Analysis of the PCNA proteins by native gel electrophoresis. Solutions containing the wild-type or mutant PCNA proteins (0.1 to 1.0 mg/ml) were run on a non-denaturing polyacrylamide gradient gel (4 to

    Journal: Biochemistry

    Article Title: Distinct structural alterations in PCNA block DNA mismatch repair †

    doi: 10.1021/bi400378e

    Figure Lengend Snippet: Trimer stability of the PCNA mutant proteins. ( A ) Analysis of the PCNA proteins by native gel electrophoresis. Solutions containing the wild-type or mutant PCNA proteins (0.1 to 1.0 mg/ml) were run on a non-denaturing polyacrylamide gradient gel (4 to

    Article Snippet: For non-denaturing polyacrylamide gel electrophoresis (PAGE), the wild-type and C22Y mutant and C81R mutant PCNA proteins (0.1 to 1.0 mg/ml) were incubated in 25 mM TrisCl, pH 7.4, 150 mM NaCl, 0.01% bromophenol blue, and 10% glycerol for 5 min and then run on a TrisCl pre-cast 4–20% gradient non-denaturing polyacrylamide gel (Bio-Rad) at 4°C at a current of 25 mA using 25 mM Tris, pH 8.3, and 0.2 M glycine running buffer.

    Techniques: Mutagenesis, Nucleic Acid Electrophoresis

    CENP-B binds to the CENP-A and H3.1 nucleosomes. ( A ) Schematic representation of CENP-B DBD binding to nucleosomes. ( B ) Electrophoretic mobility shift assay. The H3.1, CENP-A and H3.1 CATD nucleosomes (lanes 1, 3 and 5, respectively) and those complexed with the CENP-B DBD (lanes 2, 4 and 6, respectively) were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis with ethidium bromide staining. ( C ) Protein contents of the H3.1, CENP-A and H3.1 CATD nucleosomes (lanes 2, 4 and 6, respectively) and those complexed with the CENP-B DBD (lanes 3, 5 and 7, respectively), analyzed by SDS-15% polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.

    Journal: Nucleic Acids Research

    Article Title: Stable complex formation of CENP-B with the CENP-A nucleosome

    doi: 10.1093/nar/gkv405

    Figure Lengend Snippet: CENP-B binds to the CENP-A and H3.1 nucleosomes. ( A ) Schematic representation of CENP-B DBD binding to nucleosomes. ( B ) Electrophoretic mobility shift assay. The H3.1, CENP-A and H3.1 CATD nucleosomes (lanes 1, 3 and 5, respectively) and those complexed with the CENP-B DBD (lanes 2, 4 and 6, respectively) were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis with ethidium bromide staining. ( C ) Protein contents of the H3.1, CENP-A and H3.1 CATD nucleosomes (lanes 2, 4 and 6, respectively) and those complexed with the CENP-B DBD (lanes 3, 5 and 7, respectively), analyzed by SDS-15% polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.

    Article Snippet: The reconstituted nucleosomes were purified by preparative non-denaturing polyacrylamide gel electrophoresis (Prep Cell Model 491: Bio-Rad).

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Polyacrylamide Gel Electrophoresis, Staining

    CENP-B binds more stably to the proximal DNA region of the CENP-A nucleosome. ( A ) Schematic representation of the proximal and distal CENP-B box locations, relative to the CENP-A nucleosome (dotted ellipses). The upper and lower panels illustrate the nucleosomes with the 166 base-pair α-satellite DNA (used in Figures 1 and 2 ) and the 166 base-pair α-satellite (-20) DNA, respectively. ( B ) Electrophoretic mobility shift assay. The H3.1 and CENP-A nucleosomes (lanes 1, 3, 5 and 7) and those complexed with the CENP-B DBD (lanes 2, 4, 6 and 8, respectively) were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis with ethidium bromide staining. Lanes 1, 2, 5 and 6 indicate the H3.1 nucleosomes, and lanes 3, 4, 7 and 8 indicate the CENP-A nucleosomes. Lanes 1–4 and lanes 5–8 are experiments with the 166 base-pair α-satellite DNA and the 166 base-pair α-satellite (-20) DNA, respectively. ( C ) The H3.1 (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of the naked 166 base-pair α-satellite DNA. The amounts of naked 166 base-pair α-satellite DNA are 0 ng (lanes 2 and 8), 50 ng (lanes 3 and 9), 75 ng (lanes 4 and 10), 100 ng (lanes 5 and 11) and 125 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( D ) Graphic representation of the experiments shown in panel (C). The amounts (%) of H3.1 nucleosomes complexed with CENP-B DBD were plotted against the amounts of competitor DNA. Averages of three independent experiments are shown with standard deviation values. ( E ) The CENP-A nucleosomes (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of the naked 166 base-pair α-satellite DNA. The amounts of naked 166 base-pair α-satellite DNA are 0 ng (lanes 2 and 8), 50 ng (lanes 3 and 9), 75 ng (lanes 4 and 10), 100 ng (lanes 5 and 11) and 125 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( F ) Graphic representation of the experiments shown in panel (E). The amounts (%) of CENP-A nucleosomes complexed with CENP-B DBD were plotted against the amounts of competitor DNA. Averages of three independent experiments are shown with standard deviation values.

    Journal: Nucleic Acids Research

    Article Title: Stable complex formation of CENP-B with the CENP-A nucleosome

    doi: 10.1093/nar/gkv405

    Figure Lengend Snippet: CENP-B binds more stably to the proximal DNA region of the CENP-A nucleosome. ( A ) Schematic representation of the proximal and distal CENP-B box locations, relative to the CENP-A nucleosome (dotted ellipses). The upper and lower panels illustrate the nucleosomes with the 166 base-pair α-satellite DNA (used in Figures 1 and 2 ) and the 166 base-pair α-satellite (-20) DNA, respectively. ( B ) Electrophoretic mobility shift assay. The H3.1 and CENP-A nucleosomes (lanes 1, 3, 5 and 7) and those complexed with the CENP-B DBD (lanes 2, 4, 6 and 8, respectively) were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis with ethidium bromide staining. Lanes 1, 2, 5 and 6 indicate the H3.1 nucleosomes, and lanes 3, 4, 7 and 8 indicate the CENP-A nucleosomes. Lanes 1–4 and lanes 5–8 are experiments with the 166 base-pair α-satellite DNA and the 166 base-pair α-satellite (-20) DNA, respectively. ( C ) The H3.1 (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of the naked 166 base-pair α-satellite DNA. The amounts of naked 166 base-pair α-satellite DNA are 0 ng (lanes 2 and 8), 50 ng (lanes 3 and 9), 75 ng (lanes 4 and 10), 100 ng (lanes 5 and 11) and 125 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( D ) Graphic representation of the experiments shown in panel (C). The amounts (%) of H3.1 nucleosomes complexed with CENP-B DBD were plotted against the amounts of competitor DNA. Averages of three independent experiments are shown with standard deviation values. ( E ) The CENP-A nucleosomes (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of the naked 166 base-pair α-satellite DNA. The amounts of naked 166 base-pair α-satellite DNA are 0 ng (lanes 2 and 8), 50 ng (lanes 3 and 9), 75 ng (lanes 4 and 10), 100 ng (lanes 5 and 11) and 125 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( F ) Graphic representation of the experiments shown in panel (E). The amounts (%) of CENP-A nucleosomes complexed with CENP-B DBD were plotted against the amounts of competitor DNA. Averages of three independent experiments are shown with standard deviation values.

    Article Snippet: The reconstituted nucleosomes were purified by preparative non-denaturing polyacrylamide gel electrophoresis (Prep Cell Model 491: Bio-Rad).

    Techniques: Stable Transfection, Electrophoretic Mobility Shift Assay, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Standard Deviation

    Native PAGE of apolipoprotein-LPS incubations. Apolipoproteins (20 μg) were incubated with increasing amounts of LPS for 1 h and analyzed by PAGE under non-denaturing conditions. Shown are apoA-I (lane 1), incubated with 80 μg (lane 2), 130 μg (lane 3) and 180 μg LPS (lane 4). Lanes 5–8 contain chimera only (lane 5) with 80 μg (lane 6), 130 μg (lane 7) and 180 μg LPS (lane 8); lanes 9–12 contain apoLp-III (lane 9) with 80 μg (lane 10), 130 μg (lane 11) and 180 μg LPS (lane 12).

    Journal: Biochimica et biophysica acta

    Article Title: Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity

    doi: 10.1016/j.bbamem.2017.04.017

    Figure Lengend Snippet: Native PAGE of apolipoprotein-LPS incubations. Apolipoproteins (20 μg) were incubated with increasing amounts of LPS for 1 h and analyzed by PAGE under non-denaturing conditions. Shown are apoA-I (lane 1), incubated with 80 μg (lane 2), 130 μg (lane 3) and 180 μg LPS (lane 4). Lanes 5–8 contain chimera only (lane 5) with 80 μg (lane 6), 130 μg (lane 7) and 180 μg LPS (lane 8); lanes 9–12 contain apoLp-III (lane 9) with 80 μg (lane 10), 130 μg (lane 11) and 180 μg LPS (lane 12).

    Article Snippet: Proteins were incubated at 37°C for 1 h in the presence of various amounts of LPS and separated using non-denaturing PAGE (pre-cast 4–20% Tris-Glycine gels, Invitrogen, Carlsbad, CA).

    Techniques: Clear Native PAGE, Incubation, Polyacrylamide Gel Electrophoresis

    Panel A: Non-denaturing PAGE analysis showing a much greater mobility of apoLp-III compared to apoA-I and the chimera. Twenty μg of protein was electrophoresed in the absence of SDS on a 4–20% Tris-glycine gel. Lane 1: apoA-I, lane 2: apoLp-III cys /CT-apoA-I, lane 3: apoLp-III. Panel B: Size-exclusion chromatographic analysis. Protein (0.5 mg at a 1 mg/mL concentration) was applied to a Superdex-200 column. Elution of the proteins was monitored at 210 nm using a flow rate of 0.5 mL/min. ApoLp-III eluted as a single peak at 17 mL (dash-dotted line), while apoA-I (solid line) and the chimera (dotted line) elute much earlier at 11 mL.

    Journal: Biochimica et biophysica acta

    Article Title: Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity

    doi: 10.1016/j.bbamem.2017.04.017

    Figure Lengend Snippet: Panel A: Non-denaturing PAGE analysis showing a much greater mobility of apoLp-III compared to apoA-I and the chimera. Twenty μg of protein was electrophoresed in the absence of SDS on a 4–20% Tris-glycine gel. Lane 1: apoA-I, lane 2: apoLp-III cys /CT-apoA-I, lane 3: apoLp-III. Panel B: Size-exclusion chromatographic analysis. Protein (0.5 mg at a 1 mg/mL concentration) was applied to a Superdex-200 column. Elution of the proteins was monitored at 210 nm using a flow rate of 0.5 mL/min. ApoLp-III eluted as a single peak at 17 mL (dash-dotted line), while apoA-I (solid line) and the chimera (dotted line) elute much earlier at 11 mL.

    Article Snippet: Proteins were incubated at 37°C for 1 h in the presence of various amounts of LPS and separated using non-denaturing PAGE (pre-cast 4–20% Tris-Glycine gels, Invitrogen, Carlsbad, CA).

    Techniques: Polyacrylamide Gel Electrophoresis, Concentration Assay, Flow Cytometry

    Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in SDS-PAGE with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Heparanase Overexpression Reduces Hepcidin Expression, Affects Iron Homeostasis and Alters the Response to Inflammation

    doi: 10.1371/journal.pone.0164183

    Figure Lengend Snippet: Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver. (A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in SDS-PAGE with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H 2 O 2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.

    Article Snippet: Samples of 40–50 μg proteins were separated by 10–14% SDS-PAGE or 8% non-denaturing PAGE and transferred to Cellulose Nitrate Membrane (Whatman) or Hybond-P Membrane (GE).

    Techniques: Transgenic Assay, Mouse Assay, Western Blot, SDS Page, Polyacrylamide Gel Electrophoresis, Staining, Purification, Recombinant

    HepG2 cells transiently transfected with heparanase showed a reduction of hepcidin mRNA. HepG2 cells were transfected with pcDNA3.1-HPA plasmid (HPA) or empty pcDNA3.1 as control (MOCK) and harvested 48 h after the transfection. (A) Relative level of HPA mRNA was measured by qRT-PCR (B) Western blot of SDS-PAGE with anti-HPA antibodies show the levels of its latent (65 kDa) and active (50 kDa) form. Densitometry quantification of the two protein forms was performed in relation to Actin. (C) The level of hepcidin mRNA and (D) Id1 mRNA was analyzed by qPCR and normalized for Hprt1. (E) The phosphorylated (pSMAD5) and total SMAD5 were analyzed by western blot and pSMAD5 densitometry was normalized to actin. In (A) the values are expressed as–dCt for HPA mRNA, in C and D as fold change over the control (MOCK) for hepcidin and Id1 mRNA., respectively

    Journal: PLoS ONE

    Article Title: Heparanase Overexpression Reduces Hepcidin Expression, Affects Iron Homeostasis and Alters the Response to Inflammation

    doi: 10.1371/journal.pone.0164183

    Figure Lengend Snippet: HepG2 cells transiently transfected with heparanase showed a reduction of hepcidin mRNA. HepG2 cells were transfected with pcDNA3.1-HPA plasmid (HPA) or empty pcDNA3.1 as control (MOCK) and harvested 48 h after the transfection. (A) Relative level of HPA mRNA was measured by qRT-PCR (B) Western blot of SDS-PAGE with anti-HPA antibodies show the levels of its latent (65 kDa) and active (50 kDa) form. Densitometry quantification of the two protein forms was performed in relation to Actin. (C) The level of hepcidin mRNA and (D) Id1 mRNA was analyzed by qPCR and normalized for Hprt1. (E) The phosphorylated (pSMAD5) and total SMAD5 were analyzed by western blot and pSMAD5 densitometry was normalized to actin. In (A) the values are expressed as–dCt for HPA mRNA, in C and D as fold change over the control (MOCK) for hepcidin and Id1 mRNA., respectively

    Article Snippet: Samples of 40–50 μg proteins were separated by 10–14% SDS-PAGE or 8% non-denaturing PAGE and transferred to Cellulose Nitrate Membrane (Whatman) or Hybond-P Membrane (GE).

    Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, SDS Page, Real-time Polymerase Chain Reaction

    The pIgR associated IgM–ovalbumin (OVA) complexes in intestinal mucus of group 1 at 6 h after OVA challenge. (A,B) The Native-PAGE under non-reducing condition that stained by Coomassie blue and analyzed by western blot. (A) Lane 1: maker, lanes 2–7: native-PAGE result of serum and gut mucus of group 1 (2–3), group 2 (4–5), and group 3 (6–7), respectively. (B) Western blot result. Lane 1: maker, lanes 2–7: the samples of (A) reacted with anti-IgM monoclonal antibody (MAb) 2D8; lanes 8–13: the samples of (A) reacted with rabbit anti-OVA antibody; lanes 14–19: the samples of (A) reacted with mouse anti-pIgR antibody. (C) SDS-PAGE under reducing condition analyzed the samples from co-immunoprecipitation of gut mucus with rabbit anti-OVA antibody after challenge. Lane 1: maker; lane 2: protein G + rabbit anti-OVA antibody + gut mucus of group 1; lane 3: protein G + rabbit anti-OVA antibody + gut mucus of group 2; lane 4: protein G + rabbit anti-OVA antibody + gut mucus of group 3; lane 5: protein G + non-immune rabbit serum + gut mucus of group 1; lane 6: protein G + gut mucus of group 1. (D) Co-immunoprecipitation of gut mucus with anti-OVA antibody, followed by western blotting assay under reducing conditions. Lane 1: maker; lanes 2–6: Samples of (C) reacted with mouse anti-pIgR antibody; lanes 7–11: Samples of (C) reacted with anti-IgM MAb 2D8.

    Journal: Frontiers in Immunology

    Article Title: Polymeric Immunoglobulin Receptor Mediates Immune Excretion of Mucosal IgM–Antigen Complexes Across Intestinal Epithelium in Flounder (Paralichthys olivaceus)

    doi: 10.3389/fimmu.2018.01562

    Figure Lengend Snippet: The pIgR associated IgM–ovalbumin (OVA) complexes in intestinal mucus of group 1 at 6 h after OVA challenge. (A,B) The Native-PAGE under non-reducing condition that stained by Coomassie blue and analyzed by western blot. (A) Lane 1: maker, lanes 2–7: native-PAGE result of serum and gut mucus of group 1 (2–3), group 2 (4–5), and group 3 (6–7), respectively. (B) Western blot result. Lane 1: maker, lanes 2–7: the samples of (A) reacted with anti-IgM monoclonal antibody (MAb) 2D8; lanes 8–13: the samples of (A) reacted with rabbit anti-OVA antibody; lanes 14–19: the samples of (A) reacted with mouse anti-pIgR antibody. (C) SDS-PAGE under reducing condition analyzed the samples from co-immunoprecipitation of gut mucus with rabbit anti-OVA antibody after challenge. Lane 1: maker; lane 2: protein G + rabbit anti-OVA antibody + gut mucus of group 1; lane 3: protein G + rabbit anti-OVA antibody + gut mucus of group 2; lane 4: protein G + rabbit anti-OVA antibody + gut mucus of group 3; lane 5: protein G + non-immune rabbit serum + gut mucus of group 1; lane 6: protein G + gut mucus of group 1. (D) Co-immunoprecipitation of gut mucus with anti-OVA antibody, followed by western blotting assay under reducing conditions. Lane 1: maker; lanes 2–6: Samples of (C) reacted with mouse anti-pIgR antibody; lanes 7–11: Samples of (C) reacted with anti-IgM MAb 2D8.

    Article Snippet: Western Blotting and Co-Immunoprecipitation Assay To investigate the relationship of OVA, pIgR, and IgM, serum and gut mucus samples were resolved on 3–20% Native-PAGE Ready Gel (Bio-Rad) under non-reducing conditions in duplicate, one was stained with Coomassie blue R-250, the other was transferred onto PVDF membrane (Millipore, USA).

    Techniques: Clear Native PAGE, Staining, Western Blot, SDS Page, Immunoprecipitation