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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
    Analysis of mitochondrial <t>supercomplexes</t> in piriform cortex of the neuron-specific RISP KO mice treated with mitoTEMPO. Mitochondrial fraction from piriform cortex were obtained from control and RISP KO mice treated with either vehicle or with ( A ) low dose of 3 mg/kg/day or ( B ) high dose of 10 mg/kg/day of mitoTEMPO (MT). Mice were treated from weaning to 82–105 days of age. SCs were analyzed by <t>BN-PAGE</t> and western blot to detect SCs (HMW and CI+CIII), CI, CIII, CIV and CII using antibodies against NDUFA9, UQCRC1, Cox1 and SDHA subunits respectively. Low dose did not improved stability of respiratory complexes. Graphs represent the quantification of the levels of NDUFA9 ( C ) and UQCRC1 ( D ) normalized to actin in HMW SCs, CI+CIII, free CI or CIII respectively of the blots shown in ( B ). ( E ) Steady-state levels of some mitochondrial proteins in piriform cortex of vehicle and MT-treated mice by SDS-PAGE. Molecular weight (MW) for each protein is indicated in the figure. ( F ) quantification of blots in ( E ). Quantification of signal in blots was performed by densitometry analysis with ImageJ software. Protein levels were normalized to actin. Graphs represent the mean and standard deviation. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. (**) p
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    Bio-Rad preparative non denaturing polyacrylamide gel 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 <t>non-denaturing</t> 6% <t>polyacrylamide</t> <t>gel</t> <t>electrophoresis</t> 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.
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    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 <t>non-denaturing</t> 6% <t>polyacrylamide</t> <t>gel</t> <t>electrophoresis</t> 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.
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    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 <t>non-denaturing</t> 6% <t>polyacrylamide</t> <t>gel</t> <t>electrophoresis</t> 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.
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    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 <t>non-denaturing</t> 6% <t>polyacrylamide</t> <t>gel</t> <t>electrophoresis</t> 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.
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    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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    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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    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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    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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    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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    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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    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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    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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    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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    Millipore 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.
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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

    Analysis of mitochondrial supercomplexes in piriform cortex of the neuron-specific RISP KO mice treated with mitoTEMPO. Mitochondrial fraction from piriform cortex were obtained from control and RISP KO mice treated with either vehicle or with ( A ) low dose of 3 mg/kg/day or ( B ) high dose of 10 mg/kg/day of mitoTEMPO (MT). Mice were treated from weaning to 82–105 days of age. SCs were analyzed by BN-PAGE and western blot to detect SCs (HMW and CI+CIII), CI, CIII, CIV and CII using antibodies against NDUFA9, UQCRC1, Cox1 and SDHA subunits respectively. Low dose did not improved stability of respiratory complexes. Graphs represent the quantification of the levels of NDUFA9 ( C ) and UQCRC1 ( D ) normalized to actin in HMW SCs, CI+CIII, free CI or CIII respectively of the blots shown in ( B ). ( E ) Steady-state levels of some mitochondrial proteins in piriform cortex of vehicle and MT-treated mice by SDS-PAGE. Molecular weight (MW) for each protein is indicated in the figure. ( F ) quantification of blots in ( E ). Quantification of signal in blots was performed by densitometry analysis with ImageJ software. Protein levels were normalized to actin. Graphs represent the mean and standard deviation. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. (**) p

    Journal: International Journal of Molecular Sciences

    Article Title: The Organization of Mitochondrial Supercomplexes is Modulated by Oxidative Stress In Vivo in Mouse Models of Mitochondrial Encephalopathy

    doi: 10.3390/ijms19061582

    Figure Lengend Snippet: Analysis of mitochondrial supercomplexes in piriform cortex of the neuron-specific RISP KO mice treated with mitoTEMPO. Mitochondrial fraction from piriform cortex were obtained from control and RISP KO mice treated with either vehicle or with ( A ) low dose of 3 mg/kg/day or ( B ) high dose of 10 mg/kg/day of mitoTEMPO (MT). Mice were treated from weaning to 82–105 days of age. SCs were analyzed by BN-PAGE and western blot to detect SCs (HMW and CI+CIII), CI, CIII, CIV and CII using antibodies against NDUFA9, UQCRC1, Cox1 and SDHA subunits respectively. Low dose did not improved stability of respiratory complexes. Graphs represent the quantification of the levels of NDUFA9 ( C ) and UQCRC1 ( D ) normalized to actin in HMW SCs, CI+CIII, free CI or CIII respectively of the blots shown in ( B ). ( E ) Steady-state levels of some mitochondrial proteins in piriform cortex of vehicle and MT-treated mice by SDS-PAGE. Molecular weight (MW) for each protein is indicated in the figure. ( F ) quantification of blots in ( E ). Quantification of signal in blots was performed by densitometry analysis with ImageJ software. Protein levels were normalized to actin. Graphs represent the mean and standard deviation. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. (**) p

    Article Snippet: Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) Mitochondrial supercomplexes were extracted from the mitochondria enriched fraction described above using digitonin (Calbiochem) at a protein to detergent ratio of 1:8.

    Techniques: Mouse Assay, Polyacrylamide Gel Electrophoresis, Western Blot, SDS Page, Molecular Weight, Software, Standard Deviation

    Mitochondrial supercomplexes in RISP fibroblasts treated with mitoTEMPO. Control (#8) and RISP KO (#8.5) mouse fibroblasts were incubated with different concentrations of mitoTEMPO (MT) for 24 h. Cell homogenates were prepared for the analysis of ( A ) SCs by BN-PAGE and western blot to detect SCs, CI, CIII, CIV and CII using antibodies against NDUFA9, UQCRC1, Cox5b and SDHA subunits respectively or ( B ) Steady-state level of various mitochondrial proteins by SDS-PAGE and western blot. Tim23 was used as loading control for BN-PAGE and actin was used as loading control for SDS-PAGE. Asterisk and dimmed blue bar denote unrelated sample to this study (Ctrl fibroblast exposed to hypoxia). ( C , D ) Densitometry of blots in ( A , B ).

    Journal: International Journal of Molecular Sciences

    Article Title: The Organization of Mitochondrial Supercomplexes is Modulated by Oxidative Stress In Vivo in Mouse Models of Mitochondrial Encephalopathy

    doi: 10.3390/ijms19061582

    Figure Lengend Snippet: Mitochondrial supercomplexes in RISP fibroblasts treated with mitoTEMPO. Control (#8) and RISP KO (#8.5) mouse fibroblasts were incubated with different concentrations of mitoTEMPO (MT) for 24 h. Cell homogenates were prepared for the analysis of ( A ) SCs by BN-PAGE and western blot to detect SCs, CI, CIII, CIV and CII using antibodies against NDUFA9, UQCRC1, Cox5b and SDHA subunits respectively or ( B ) Steady-state level of various mitochondrial proteins by SDS-PAGE and western blot. Tim23 was used as loading control for BN-PAGE and actin was used as loading control for SDS-PAGE. Asterisk and dimmed blue bar denote unrelated sample to this study (Ctrl fibroblast exposed to hypoxia). ( C , D ) Densitometry of blots in ( A , B ).

    Article Snippet: Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) Mitochondrial supercomplexes were extracted from the mitochondria enriched fraction described above using digitonin (Calbiochem) at a protein to detergent ratio of 1:8.

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, SDS Page

    Blue native gel electrophoresis analysis of mitochondrial supercomplexes in brain regions of neuron-specific COX10 and RISP KO mice. Mitochondrial fraction was isolated from the different brain regions of control and KO mice at different ages (78–90 days). Mitochondrial proteins were extracted with digitonin and separated by blue native gel electrophoresis followed by western blot. CI, CIII and CIV were analyzed using NDUFA9, UQCRC1 and Cox1 antibodies respectively. Tim23 was used as mitochondrial loading control. Mitochondrial supercomplexes are indicated in the figure as SC (HMW and CI+CIII arrangements) in addition to free CI, free CIII and the monomer of CIV. Mitochondrial proteins were extracted from ( A ) hippocampus, ( D ) cingulate cortex and ( G ) piriform cortex from control and KO mice at ages indicated. ( B , C , E , F , H , I ) Graphs represent mean and standard deviation of levels of NDUFA9 normalized to Tim23 and expressed as percentage of total signal for hippocampus of COX10 ( B ) and RISP ( C ); cingulate cortex of COX10 ( E ) and RISP ( F ) and piriform cortex of COX10 ( H ) and RISP ( I ) mice. Antibody signals were quantified by densitometry of blots using ImageJ. (*) p

    Journal: International Journal of Molecular Sciences

    Article Title: The Organization of Mitochondrial Supercomplexes is Modulated by Oxidative Stress In Vivo in Mouse Models of Mitochondrial Encephalopathy

    doi: 10.3390/ijms19061582

    Figure Lengend Snippet: Blue native gel electrophoresis analysis of mitochondrial supercomplexes in brain regions of neuron-specific COX10 and RISP KO mice. Mitochondrial fraction was isolated from the different brain regions of control and KO mice at different ages (78–90 days). Mitochondrial proteins were extracted with digitonin and separated by blue native gel electrophoresis followed by western blot. CI, CIII and CIV were analyzed using NDUFA9, UQCRC1 and Cox1 antibodies respectively. Tim23 was used as mitochondrial loading control. Mitochondrial supercomplexes are indicated in the figure as SC (HMW and CI+CIII arrangements) in addition to free CI, free CIII and the monomer of CIV. Mitochondrial proteins were extracted from ( A ) hippocampus, ( D ) cingulate cortex and ( G ) piriform cortex from control and KO mice at ages indicated. ( B , C , E , F , H , I ) Graphs represent mean and standard deviation of levels of NDUFA9 normalized to Tim23 and expressed as percentage of total signal for hippocampus of COX10 ( B ) and RISP ( C ); cingulate cortex of COX10 ( E ) and RISP ( F ) and piriform cortex of COX10 ( H ) and RISP ( I ) mice. Antibody signals were quantified by densitometry of blots using ImageJ. (*) p

    Article Snippet: Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) Mitochondrial supercomplexes were extracted from the mitochondria enriched fraction described above using digitonin (Calbiochem) at a protein to detergent ratio of 1:8.

    Techniques: Nucleic Acid Electrophoresis, Mouse Assay, Isolation, Western Blot, Standard Deviation

    Demonstration of differential release of PF4 from complexes formed with branded and US-generic enoxaparins. A, Chromatogram of SE-FPLC analysis of PF4 alone and all Lot 1, Batch 1 PF4–enoxaparin complexes performed at 0.15 mol/L NaCl (upper panel) and 0.75 mol/L NaCl (lower panel). B, 4% to 20% gradient native PAGE gel analysis of PF4–enoxaparin complexes under nonreducing conditions followed by silver staining to resolve the protein bands. These data are representative of 3 independent analyses. ELISA indicates enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PF4, platelet factor 4; PF4–H, PF4–heparin complex; SD, standard deviation; SE-FPLC, size exclusion-fast performance liquid chromatography.

    Journal: Clinical and Applied Thrombosis/Hemostasis

    Article Title: Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model

    doi: 10.1177/1076029614562037

    Figure Lengend Snippet: Demonstration of differential release of PF4 from complexes formed with branded and US-generic enoxaparins. A, Chromatogram of SE-FPLC analysis of PF4 alone and all Lot 1, Batch 1 PF4–enoxaparin complexes performed at 0.15 mol/L NaCl (upper panel) and 0.75 mol/L NaCl (lower panel). B, 4% to 20% gradient native PAGE gel analysis of PF4–enoxaparin complexes under nonreducing conditions followed by silver staining to resolve the protein bands. These data are representative of 3 independent analyses. ELISA indicates enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PF4, platelet factor 4; PF4–H, PF4–heparin complex; SD, standard deviation; SE-FPLC, size exclusion-fast performance liquid chromatography.

    Article Snippet: Thereafter, 25 ng of the complexes were separated by 4% to 20% gradient native polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions and visualized by silver staining (ProteoSilver Silver Stain Kit, Sigma-Aldrich) against molecular weight standards.

    Techniques: Fast Protein Liquid Chromatography, Clear Native PAGE, Silver Staining, Enzyme-linked Immunosorbent Assay, Polyacrylamide Gel Electrophoresis, Standard Deviation, Liquid Chromatography

    CD spectra of Protein A-binding aptamer variants and electrophoretic results. (a) The representative CD spectra of DNA aptamer PA#2/8 (350 nM), native Protein A (230 μg/mL) and aptamer-protein-complex (PA#2/8-Protein A) are represented by black, red and blue line, respectively. The arithmetic sum of PA#2/8 and Protein A is represented by a dashed green line. The difference spectrum between complex and arithmetic sum is represented by a yellow line. The aptamer variant PA#2/8[S28-50] truncated at both ends was used as negative control. In this case, the difference between arithmetic sum and complex shows no detectable signal (purple line). (b) CD spectra of different Protein A-binding aptamer variants in modified 25 mM Britton-Robinson buffer (pH 7.6) in the presence of 100 mM NaCl, 10 mM MgCl 2 , 1 mM CaCl 2 and 5 mM KCl were compared (PA#2/8, black line; PA#2/8[S1-58], green line; PA#2/8[S1-50], red line and PA#2/8[S1-43], blue line). The aptamers were used at a concentration of 350 nM. (c) The corresponding CD melting curves obtained at 265 nm are depicted. (d) Separation of different aptamer variants by native gel electrophoresis and visualisation by StainsAll staining. The mobilities of molecular standards (mix of d(AC) 9 + d(AC) 14 + d(AC) 18 , mix of d(AC) 26 + d(AC) 36 , d(G 3 T 2 A) 3 G 3 ) and d(G 3 T 2 A) 7 G 3 T 14 ) are shown on the gel at the right. The positions of the standards are additionally indicated as arrows on the left including their sizes in nt. G-quadruplex forming standards are highlighted in red. Electrophoretic separation was performed in a 10% polyacrylamide gel in 25 mM Britton-Robinson buffer (pH 7.6) and 100 mM NaCl, 10 mM MgCl 2 and 5 mM KCl at 37 °C. The loading buffer also contained 1 mM CaCl 2 and 0.005% Tween 20.

    Journal: Scientific Reports

    Article Title: G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA

    doi: 10.1038/srep33812

    Figure Lengend Snippet: CD spectra of Protein A-binding aptamer variants and electrophoretic results. (a) The representative CD spectra of DNA aptamer PA#2/8 (350 nM), native Protein A (230 μg/mL) and aptamer-protein-complex (PA#2/8-Protein A) are represented by black, red and blue line, respectively. The arithmetic sum of PA#2/8 and Protein A is represented by a dashed green line. The difference spectrum between complex and arithmetic sum is represented by a yellow line. The aptamer variant PA#2/8[S28-50] truncated at both ends was used as negative control. In this case, the difference between arithmetic sum and complex shows no detectable signal (purple line). (b) CD spectra of different Protein A-binding aptamer variants in modified 25 mM Britton-Robinson buffer (pH 7.6) in the presence of 100 mM NaCl, 10 mM MgCl 2 , 1 mM CaCl 2 and 5 mM KCl were compared (PA#2/8, black line; PA#2/8[S1-58], green line; PA#2/8[S1-50], red line and PA#2/8[S1-43], blue line). The aptamers were used at a concentration of 350 nM. (c) The corresponding CD melting curves obtained at 265 nm are depicted. (d) Separation of different aptamer variants by native gel electrophoresis and visualisation by StainsAll staining. The mobilities of molecular standards (mix of d(AC) 9 + d(AC) 14 + d(AC) 18 , mix of d(AC) 26 + d(AC) 36 , d(G 3 T 2 A) 3 G 3 ) and d(G 3 T 2 A) 7 G 3 T 14 ) are shown on the gel at the right. The positions of the standards are additionally indicated as arrows on the left including their sizes in nt. G-quadruplex forming standards are highlighted in red. Electrophoretic separation was performed in a 10% polyacrylamide gel in 25 mM Britton-Robinson buffer (pH 7.6) and 100 mM NaCl, 10 mM MgCl 2 and 5 mM KCl at 37 °C. The loading buffer also contained 1 mM CaCl 2 and 0.005% Tween 20.

    Article Snippet: Electrophoresis Native polyacrylamide gel electrophoresis (PAGE) was performed in a temperature-controlled vertical electrophoretic apparatus (Z375039-1EA; Sigma-Aldrich, San Francisco, CA).

    Techniques: Binding Assay, Variant Assay, Negative Control, Modification, Concentration Assay, Nucleic Acid Electrophoresis, Staining

    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

    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