horseradish peroxidase conjugated streptavidin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Characterization of CPMV labeling with the biotinylated R5 peptide. (A) Size exclusion chromatography of wild-type CPMV, CPMV–R5L and CPMV–R5Hat 280 nm. (B) ECL dot blot of purified CPMV particles. The number of biotin labels per particle was determined using standardized biotin concentrations and Chemidoc XRS software. (C) Native gel electrophoresis of intact CPMV particles (10 µg) using a 0.8% (w/v) agarose gel. Particles were visualized under UV light. Lane 1 = CPMV, 2 = CPMV–4FB, 3 = CPMV–R5H, 4 = CPMV–PFB, 5 = CPMV–R5L. (D) SDS–PAGE of CPMV particles (10 µg) using a 4–12% Bis-Tris gel and western blotting using <t>streptavidin–alkaline</t> phosphatase to detect the N-terminal biotin tag of the R5 peptide. (E) Zeta potential of CPMV wild type, CPMV–R5L and CPMV–R5H formulations.
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Development of viral nanoparticles for efficient intracellular delivery †"

    Article Title: Development of viral nanoparticles for efficient intracellular delivery †

    Journal: Nanoscale

    doi: 10.1039/c2nr30366c

    Characterization of CPMV labeling with the biotinylated R5 peptide. (A) Size exclusion chromatography of wild-type CPMV, CPMV–R5L and CPMV–R5Hat 280 nm. (B) ECL dot blot of purified CPMV particles. The number of biotin labels per particle was determined using standardized biotin concentrations and Chemidoc XRS software. (C) Native gel electrophoresis of intact CPMV particles (10 µg) using a 0.8% (w/v) agarose gel. Particles were visualized under UV light. Lane 1 = CPMV, 2 = CPMV–4FB, 3 = CPMV–R5H, 4 = CPMV–PFB, 5 = CPMV–R5L. (D) SDS–PAGE of CPMV particles (10 µg) using a 4–12% Bis-Tris gel and western blotting using streptavidin–alkaline phosphatase to detect the N-terminal biotin tag of the R5 peptide. (E) Zeta potential of CPMV wild type, CPMV–R5L and CPMV–R5H formulations.
    Figure Legend Snippet: Characterization of CPMV labeling with the biotinylated R5 peptide. (A) Size exclusion chromatography of wild-type CPMV, CPMV–R5L and CPMV–R5Hat 280 nm. (B) ECL dot blot of purified CPMV particles. The number of biotin labels per particle was determined using standardized biotin concentrations and Chemidoc XRS software. (C) Native gel electrophoresis of intact CPMV particles (10 µg) using a 0.8% (w/v) agarose gel. Particles were visualized under UV light. Lane 1 = CPMV, 2 = CPMV–4FB, 3 = CPMV–R5H, 4 = CPMV–PFB, 5 = CPMV–R5L. (D) SDS–PAGE of CPMV particles (10 µg) using a 4–12% Bis-Tris gel and western blotting using streptavidin–alkaline phosphatase to detect the N-terminal biotin tag of the R5 peptide. (E) Zeta potential of CPMV wild type, CPMV–R5L and CPMV–R5H formulations.

    Techniques Used: Labeling, Size-exclusion Chromatography, Dot Blot, Purification, Software, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, SDS Page, Western Blot

    2) Product Images from "Mouse Bestrophin-2 Is a Bona fide Cl− Channel"

    Article Title: Mouse Bestrophin-2 Is a Bona fide Cl− Channel

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200409031

    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: streptavidin labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).
    Figure Legend Snippet: Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: streptavidin labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).

    Techniques Used: Labeling, Immunoprecipitation, Transfection, SDS Page, Western Blot, Incubation

    3) Product Images from "Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation"

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000666

    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Figure Legend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Techniques Used: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.
    Figure Legend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Techniques Used: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P
    Figure Legend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Techniques Used: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P
    Figure Legend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Techniques Used: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P
    Figure Legend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Techniques Used: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    4) Product Images from "Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis"

    Article Title: Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis

    Journal: Journal of Proteome Research

    doi: 10.1021/pr200918f

    Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).
    Figure Legend Snippet: Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Techniques Used: Modification, Transgenic Assay, Expressing, FLAG-tag, Labeling, Protein Extraction, Generated, Affinity Purification, SDS Page, Recombinant

    5) Product Images from "Biotinylation is a natural, albeit rare, modification of human histones"

    Article Title: Biotinylation is a natural, albeit rare, modification of human histones

    Journal: Molecular genetics and metabolism

    doi: 10.1016/j.ymgme.2011.08.030

    Comparison of histone extraction protocols, and specificity testing of streptavidin and anti-biotin Panel A: Nuclear histones were extracted from Jurkat cells (lanes 1, 5, and 9) or HeLa cells (lanes 3, 7, and 11) by using HCl; for comparison histones were extracted from Jurkat cells (lanes 2, 6, and 10) or HeLa cells (lanes 4, 8, and 12) by using H 2 SO 4 +TCA+acetone/HCl+acetone. Histones were probed with coomassie blue (lanes 1–4) and antibodies to the C-termini in histone H3 (lanes 5–8) and H4 (lanes 9–12). Panel B: HCl extracts of Jurkat cell histones (lanes 1, 4, 7, 10, and 13), recombinant human histone H4 (lanes 2, 5, 8, 11, and 14), and chemically biotinylated histone H4 (lanes 3, 6, 9, 12, and 15) were probed with streptavidin without biotin competitor (lanes 1–3) and with 5 mM free biotin (lanes 4–6), and with anti-biotin without biotin competitor (lanes 7–9) and with 5 mM free biotin (lanes 10–12), and with coomassie blue (lanes 13–15).
    Figure Legend Snippet: Comparison of histone extraction protocols, and specificity testing of streptavidin and anti-biotin Panel A: Nuclear histones were extracted from Jurkat cells (lanes 1, 5, and 9) or HeLa cells (lanes 3, 7, and 11) by using HCl; for comparison histones were extracted from Jurkat cells (lanes 2, 6, and 10) or HeLa cells (lanes 4, 8, and 12) by using H 2 SO 4 +TCA+acetone/HCl+acetone. Histones were probed with coomassie blue (lanes 1–4) and antibodies to the C-termini in histone H3 (lanes 5–8) and H4 (lanes 9–12). Panel B: HCl extracts of Jurkat cell histones (lanes 1, 4, 7, 10, and 13), recombinant human histone H4 (lanes 2, 5, 8, 11, and 14), and chemically biotinylated histone H4 (lanes 3, 6, 9, 12, and 15) were probed with streptavidin without biotin competitor (lanes 1–3) and with 5 mM free biotin (lanes 4–6), and with anti-biotin without biotin competitor (lanes 7–9) and with 5 mM free biotin (lanes 10–12), and with coomassie blue (lanes 13–15).

    Techniques Used: Recombinant

    Biotinylation marks can be detected in bulk extracts from human cells, using streptavidin and anti-biotin as probes Bulk extracts of histone extracts from various cell lineages were probed with streptavidin (panel A), anti-biotin (panel B), and the loading and transfer controls coomassie blue (panel C), anti-H3 (panel D), and anti-H4 (panel E). Panel F: Histones from HeLa cells were extracted with H 2 SO 4 +TCA+acetone/HCl+acetone. Ten or five microgram of histones were loaded per well. Blots were blocked with PBS containing 5% BSA. After probing with horseradish peroxidase-conjugated anti-biotin or Nutravidin, the blots were washed for 6 hrs, and exposed to autoradiography film for 1 min.
    Figure Legend Snippet: Biotinylation marks can be detected in bulk extracts from human cells, using streptavidin and anti-biotin as probes Bulk extracts of histone extracts from various cell lineages were probed with streptavidin (panel A), anti-biotin (panel B), and the loading and transfer controls coomassie blue (panel C), anti-H3 (panel D), and anti-H4 (panel E). Panel F: Histones from HeLa cells were extracted with H 2 SO 4 +TCA+acetone/HCl+acetone. Ten or five microgram of histones were loaded per well. Blots were blocked with PBS containing 5% BSA. After probing with horseradish peroxidase-conjugated anti-biotin or Nutravidin, the blots were washed for 6 hrs, and exposed to autoradiography film for 1 min.

    Techniques Used: Autoradiography

    Specificity of antibodies to H3K4bio, H3K9bio and H3K18bio Panel A: Confirmation of equal loading of peptides H3K4bio, H3K9bio, and H3K18bio with streptavidin. Panel B: Transblots of peptides N 1–25 (non-biotinylated negative control), H3K4bio, H3K9bio, and H3K18bio were probed with anti-H3K4bio (left), anti-H3K9bio (middle), and anti-H3K18bio (right). Panel C: HCl extracts of histones from Jurkat cells were probed using streptavidin, anti-H3K9bio, and anti-H3K18bio; samples of biotin-free histones (“-”) were generated by using avidin agarose. Panel D: Bulk HCl extracts of histones from Jurkat cells were probed with anti-H3K9bio (top) and anti-H3K18bio (bottom) after pre-incubation of antibodies with increasing amounts of competing peptides H3K4bio, H3K9bio, and H3K18bio; controls (“C”) were prepared without peptide competitors. Note the difference in the order of peptide competitors in the two gels. For some gels, bands from the same analytical runs were electronically re-arranged to facilitate comparisons. Panel E: Peptides H3K9bio, H3K9ac, and H3K9me2 were probed with streptavidin (lanes 1, 2, 9 and 10), anti-H3K9bio (lanes 3, 4, 11 and 12), anti-H3K9ac (lanes 5 and 6), and anti-H3K9me2 (lanes 13 and 14); Ponceau S was used as loading control (lanes 7, 8, 15 and 16). Panel F: Peptides H3K18bio and H3K18ac were probed with streptavidin (lanes 1 and 2), anti-H3K18bio (lanes 3 and 4) and anti-H3K18ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8).
    Figure Legend Snippet: Specificity of antibodies to H3K4bio, H3K9bio and H3K18bio Panel A: Confirmation of equal loading of peptides H3K4bio, H3K9bio, and H3K18bio with streptavidin. Panel B: Transblots of peptides N 1–25 (non-biotinylated negative control), H3K4bio, H3K9bio, and H3K18bio were probed with anti-H3K4bio (left), anti-H3K9bio (middle), and anti-H3K18bio (right). Panel C: HCl extracts of histones from Jurkat cells were probed using streptavidin, anti-H3K9bio, and anti-H3K18bio; samples of biotin-free histones (“-”) were generated by using avidin agarose. Panel D: Bulk HCl extracts of histones from Jurkat cells were probed with anti-H3K9bio (top) and anti-H3K18bio (bottom) after pre-incubation of antibodies with increasing amounts of competing peptides H3K4bio, H3K9bio, and H3K18bio; controls (“C”) were prepared without peptide competitors. Note the difference in the order of peptide competitors in the two gels. For some gels, bands from the same analytical runs were electronically re-arranged to facilitate comparisons. Panel E: Peptides H3K9bio, H3K9ac, and H3K9me2 were probed with streptavidin (lanes 1, 2, 9 and 10), anti-H3K9bio (lanes 3, 4, 11 and 12), anti-H3K9ac (lanes 5 and 6), and anti-H3K9me2 (lanes 13 and 14); Ponceau S was used as loading control (lanes 7, 8, 15 and 16). Panel F: Peptides H3K18bio and H3K18ac were probed with streptavidin (lanes 1 and 2), anti-H3K18bio (lanes 3 and 4) and anti-H3K18ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8).

    Techniques Used: Negative Control, Generated, Avidin-Biotin Assay, Incubation

    Specificity of antibodies to H4K8bio and H4K12bio Panel A: Transblots of peptides N 1–19 (non-biotinylated negative control), H4K8bio, and H4K12bio were probed with anti-H4K8bio; pre-immune serum was used as negative control. Panel B: HCl extracts of histones from Jurkat cells were probed using streptavidin and anti-H4K8bio; samples of biotin-free histones (“-”) were generated by using avidin agarose. Panel C: HCl extracts of histones from Jurkat cells were probed with anti-H4K8bio after pre-incubation of antibodies with increasing amounts of competing peptides H4K8bio and H4K12bio; controls (“C”) were prepared without peptide competitors. For some gels, bands from the same analytical runs were electronically rearranged to facilitate comparisons. Panel D: Peptides H4K8bio and H4K8ac were probed with streptavidin (lanes 1 and 2), anti-H4K8bio (lanes 3 and 4) and anti-H4K8ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8). Panel E: Peptides H4K12bio and H4K12ac were probed with streptavidin (lanes 1 and 2), anti-H4K12bio (lanes 3 and 4) and anti-H4K12ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8).
    Figure Legend Snippet: Specificity of antibodies to H4K8bio and H4K12bio Panel A: Transblots of peptides N 1–19 (non-biotinylated negative control), H4K8bio, and H4K12bio were probed with anti-H4K8bio; pre-immune serum was used as negative control. Panel B: HCl extracts of histones from Jurkat cells were probed using streptavidin and anti-H4K8bio; samples of biotin-free histones (“-”) were generated by using avidin agarose. Panel C: HCl extracts of histones from Jurkat cells were probed with anti-H4K8bio after pre-incubation of antibodies with increasing amounts of competing peptides H4K8bio and H4K12bio; controls (“C”) were prepared without peptide competitors. For some gels, bands from the same analytical runs were electronically rearranged to facilitate comparisons. Panel D: Peptides H4K8bio and H4K8ac were probed with streptavidin (lanes 1 and 2), anti-H4K8bio (lanes 3 and 4) and anti-H4K8ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8). Panel E: Peptides H4K12bio and H4K12ac were probed with streptavidin (lanes 1 and 2), anti-H4K12bio (lanes 3 and 4) and anti-H4K12ac (lanes 5 and 6); Ponceau S was used as loading control (lanes 7 and 8).

    Techniques Used: Negative Control, Generated, Avidin-Biotin Assay, Incubation

    Validation of the biotin depletion and repletion protocol Panel A: Jurkat cells after a 2-wk depletion in biotin-deficient medium (0.025 nM, lane 1) compared with cells cultured in medium containing a physiological concentration of biotin (0.25 nM) (lane 2), and cells after a 1-wk repletion in medium containing a pharmacological concentration of biotin (10 nM) (lane 3). Biotinylated carboxylases were probed using streptavidin (SA). Equal expression, loading, and transfer of carboxylases was confirmed using anti-PC and anti-PCC. Panel B: As described for panel A, but HeLa cells (lanes 4–6) were substituted for Jurkat cells.
    Figure Legend Snippet: Validation of the biotin depletion and repletion protocol Panel A: Jurkat cells after a 2-wk depletion in biotin-deficient medium (0.025 nM, lane 1) compared with cells cultured in medium containing a physiological concentration of biotin (0.25 nM) (lane 2), and cells after a 1-wk repletion in medium containing a pharmacological concentration of biotin (10 nM) (lane 3). Biotinylated carboxylases were probed using streptavidin (SA). Equal expression, loading, and transfer of carboxylases was confirmed using anti-PC and anti-PCC. Panel B: As described for panel A, but HeLa cells (lanes 4–6) were substituted for Jurkat cells.

    Techniques Used: Cell Culture, Concentration Assay, Expressing, Periodic Counter-current Chromatography

    6) Product Images from "Acetylation and activation of STAT3 mediated by nuclear translocation of CD44"

    Article Title: Acetylation and activation of STAT3 mediated by nuclear translocation of CD44

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200812060

    Nuclear CD44 associates with STAT3 and functions to modulate transcription. (A) Confocal microscopy of H1299 cells cultured in serum-free medium for 24 h and treated with control IgG or H-3 for 1 h. (B) Nuclear (N) and cytosolic (C) fractions were prepared from AZ521/mock and AZ521/CD44 cells and immunoprecipitated followed by Western blotting. (C) Nuclear (Nuc) and cytosolic (Cyto) fractions were immunoprecipitated followed by Western blotting. (D) Nuclear extracts were prepared from the parental H1299 and HT29 cells or cell clones stably harboring lentivirus-encoded control (Cont) shRNA or shRNA targeting CD44 and immunoprecipitated followed by Western blotting. (E) Confocal microscopy of AZ521/CD44 cells with anti-STAT3 (in red) after incubation with control IgG or H-3 for 1 and 2.5 h. (F) AZ521/CD44 cells were incubated with biotin-conjugated H-3 at 4°C. After removal to 37°C for 1 h, cytosolic and nuclear fractions and immunoprecipitates from streptavidin beads were analyzed by Western blotting. (G) Nuclear extracts were prepared from AZ521/CD44 (top) and AZ521/CD44(NLS) mutant (bottom) cells after cross-linking with 1% formaldehyde. ChIP was performed using anti-CD44 and anti-STAT3. PCR amplification of designated regions within cyclin D1 promoter was performed. (H) Nuclear extracts were prepared from AZ521/mock and AZ521/CD44 s cells cultured in serum-free medium for 24 h. EMSA was performed with biotin-labeled double-stranded oligonucleotide probes corresponding to various regions of cyclin D1 promoter in the presence and absence of anti-CD44, anti-STAT3, or anti-p300 antibodies. Shifted and supershifted complexes are indicated by arrowheads. A nonspecific band is indicated by an asterisk. Ab, antibody. (I) Reporter assays were performed in AZ521 cells and AZ521 cell clones stably harboring lentivirus-encoded control shRNA or shRNA targeting STAT3 or p300 using the pFR-Luc reporter plasmid containing five copies of GAL4 DNA–binding sites. Data are presented as the means ± SD and were derived from at least three independent experiments. *, P
    Figure Legend Snippet: Nuclear CD44 associates with STAT3 and functions to modulate transcription. (A) Confocal microscopy of H1299 cells cultured in serum-free medium for 24 h and treated with control IgG or H-3 for 1 h. (B) Nuclear (N) and cytosolic (C) fractions were prepared from AZ521/mock and AZ521/CD44 cells and immunoprecipitated followed by Western blotting. (C) Nuclear (Nuc) and cytosolic (Cyto) fractions were immunoprecipitated followed by Western blotting. (D) Nuclear extracts were prepared from the parental H1299 and HT29 cells or cell clones stably harboring lentivirus-encoded control (Cont) shRNA or shRNA targeting CD44 and immunoprecipitated followed by Western blotting. (E) Confocal microscopy of AZ521/CD44 cells with anti-STAT3 (in red) after incubation with control IgG or H-3 for 1 and 2.5 h. (F) AZ521/CD44 cells were incubated with biotin-conjugated H-3 at 4°C. After removal to 37°C for 1 h, cytosolic and nuclear fractions and immunoprecipitates from streptavidin beads were analyzed by Western blotting. (G) Nuclear extracts were prepared from AZ521/CD44 (top) and AZ521/CD44(NLS) mutant (bottom) cells after cross-linking with 1% formaldehyde. ChIP was performed using anti-CD44 and anti-STAT3. PCR amplification of designated regions within cyclin D1 promoter was performed. (H) Nuclear extracts were prepared from AZ521/mock and AZ521/CD44 s cells cultured in serum-free medium for 24 h. EMSA was performed with biotin-labeled double-stranded oligonucleotide probes corresponding to various regions of cyclin D1 promoter in the presence and absence of anti-CD44, anti-STAT3, or anti-p300 antibodies. Shifted and supershifted complexes are indicated by arrowheads. A nonspecific band is indicated by an asterisk. Ab, antibody. (I) Reporter assays were performed in AZ521 cells and AZ521 cell clones stably harboring lentivirus-encoded control shRNA or shRNA targeting STAT3 or p300 using the pFR-Luc reporter plasmid containing five copies of GAL4 DNA–binding sites. Data are presented as the means ± SD and were derived from at least three independent experiments. *, P

    Techniques Used: Confocal Microscopy, Cell Culture, Immunoprecipitation, Western Blot, Clone Assay, Stable Transfection, shRNA, Incubation, Mutagenesis, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Labeling, Plasmid Preparation, Binding Assay, Derivative Assay

    Nuclear localization of full-length CD44. (A) Immunohistochemistry of nuclear CD44 in human gastric mucosal (a) and cancerous (b–d) tissues using anti-CD44 v 6 antibody, with nuclei counterstained in hematoxylin. (B) Western blot analyses of nuclear (Nuc) and cytosolic (Cyto) fractions of human gastric (GC), colon (CRC), and lung (NSCLC) cancer cell lines. NUGC, Nagoya University gastric cancer. (C and D) HT29 (C) and H1299 (D) cells were surface labeled with biotin at 4°C followed by incubation at 37°C in the presence of OPN (C) and H-3 (D). The nuclear fraction was incubated with streptavidin beads and subjected to Western blotting. (B–D) Molecular mass is shown in kilodaltons. (E) H1299 and AZ521/CD44 cells were suspended in medium for 30 min, replated on dishes for 1 h, and immunostained using H-3 (top) and anti-myc (bottom) antibodies. Representative images taken by confocal laser microscopy are shown. (F) H1299 cells were incubated with biotin-conjugated control IgG or H-3 at 4°C followed by further incubation at 37°C for 60 min. After removing surface-retained biotin, cells were fixed and stained by avidin. Bars: (A) 100 µm; (E) 10 µm; (F) 25 µm.
    Figure Legend Snippet: Nuclear localization of full-length CD44. (A) Immunohistochemistry of nuclear CD44 in human gastric mucosal (a) and cancerous (b–d) tissues using anti-CD44 v 6 antibody, with nuclei counterstained in hematoxylin. (B) Western blot analyses of nuclear (Nuc) and cytosolic (Cyto) fractions of human gastric (GC), colon (CRC), and lung (NSCLC) cancer cell lines. NUGC, Nagoya University gastric cancer. (C and D) HT29 (C) and H1299 (D) cells were surface labeled with biotin at 4°C followed by incubation at 37°C in the presence of OPN (C) and H-3 (D). The nuclear fraction was incubated with streptavidin beads and subjected to Western blotting. (B–D) Molecular mass is shown in kilodaltons. (E) H1299 and AZ521/CD44 cells were suspended in medium for 30 min, replated on dishes for 1 h, and immunostained using H-3 (top) and anti-myc (bottom) antibodies. Representative images taken by confocal laser microscopy are shown. (F) H1299 cells were incubated with biotin-conjugated control IgG or H-3 at 4°C followed by further incubation at 37°C for 60 min. After removing surface-retained biotin, cells were fixed and stained by avidin. Bars: (A) 100 µm; (E) 10 µm; (F) 25 µm.

    Techniques Used: Immunohistochemistry, Western Blot, Labeling, Incubation, Microscopy, Staining, Avidin-Biotin Assay

    7) Product Images from "Peptide-based sequestration of the adaptor protein Nck1 in pancreatic β cells enhances insulin biogenesis and protects against diabetogenic stresses"

    Article Title: Peptide-based sequestration of the adaptor protein Nck1 in pancreatic β cells enhances insulin biogenesis and protects against diabetogenic stresses

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002728

    TAT-Tyr(P) 561 binds to Nck. A , alignment of the PERK juxtamembrane sequence surrounding the tyrosine residue involved in binding Nck and related synthetic peptides conjugated to the TAT protein-transduction domain ( PTD ). B , TAT-Tyr(P) 561 binding recombinant GST proteins in vitro as revealed by Western blotting ( WB ) with PERK-Tyr(P) 561 antibody ( n = 2 independent experiments). SH2M , SH2 domain mutant. GST Western blots show GST–Nck proteins ( top ) and GST and GST–Nck degradation products ( bottom ). C , Nck pulldown using biotin–TAT-Tyr(P) 561 immobilized on streptavidin beads and MIN6 or INS-1 cell lysates. Nck was detected by Western blotting with a pan-Nck antibody. The ability of biotin–TAT-Tyr(P) 561 to bind lysate-derived Nck was confirmed in at least five independent experiments. D , Nck pulldown using biotin–TAT-Tyr(P) 561 or biotin–TAT-Phe immobilized on streptavidin beads and MIN6 cell lysate as described above ( n = 2 independent experiments).
    Figure Legend Snippet: TAT-Tyr(P) 561 binds to Nck. A , alignment of the PERK juxtamembrane sequence surrounding the tyrosine residue involved in binding Nck and related synthetic peptides conjugated to the TAT protein-transduction domain ( PTD ). B , TAT-Tyr(P) 561 binding recombinant GST proteins in vitro as revealed by Western blotting ( WB ) with PERK-Tyr(P) 561 antibody ( n = 2 independent experiments). SH2M , SH2 domain mutant. GST Western blots show GST–Nck proteins ( top ) and GST and GST–Nck degradation products ( bottom ). C , Nck pulldown using biotin–TAT-Tyr(P) 561 immobilized on streptavidin beads and MIN6 or INS-1 cell lysates. Nck was detected by Western blotting with a pan-Nck antibody. The ability of biotin–TAT-Tyr(P) 561 to bind lysate-derived Nck was confirmed in at least five independent experiments. D , Nck pulldown using biotin–TAT-Tyr(P) 561 or biotin–TAT-Phe immobilized on streptavidin beads and MIN6 cell lysate as described above ( n = 2 independent experiments).

    Techniques Used: Sequencing, Binding Assay, Transduction, Recombinant, In Vitro, Western Blot, Mutagenesis, Derivative Assay

    8) Product Images from "Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites"

    Article Title: Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-6-66

    Western blot analysis of biotinylated P. chabaudi PESAs . P. chabaudi infected erythrocytes were obtained from two infected CBA mice by heart puncture and cultured for 6 hours to allow ring stage parasites to mature. Panel A . Western blotting of P. chabaudi infected erythrocyte membrane extracts prepared by osmotic lysis. Biotinylated proteins were detected using horseradish peroxidase-conjugated streptavidin. Solid arrowheads highlight infected erythrocyte-specific biotinylated proteins. Panel B . Silver stained duplicate gel of that shown in Panel A to confirm the cell surface specificity of trypsinization. U : uninfected erythrocyte, I : infected erythrocyte.
    Figure Legend Snippet: Western blot analysis of biotinylated P. chabaudi PESAs . P. chabaudi infected erythrocytes were obtained from two infected CBA mice by heart puncture and cultured for 6 hours to allow ring stage parasites to mature. Panel A . Western blotting of P. chabaudi infected erythrocyte membrane extracts prepared by osmotic lysis. Biotinylated proteins were detected using horseradish peroxidase-conjugated streptavidin. Solid arrowheads highlight infected erythrocyte-specific biotinylated proteins. Panel B . Silver stained duplicate gel of that shown in Panel A to confirm the cell surface specificity of trypsinization. U : uninfected erythrocyte, I : infected erythrocyte.

    Techniques Used: Western Blot, Infection, Crocin Bleaching Assay, Mouse Assay, Cell Culture, Lysis, Staining

    Detergent and osmotic lysis extracts of biotinylated P. falciparum PESAs . Panel A . Western blotting of detergent extracts from surface biotinylated P. falciparum infected erythrocytes. Biotinylated proteins were detected by horseradish peroxidase-linked streptavidin. Panel B . Western blotting of surface biotinylated P. falciparum infected erythrocyte extracts prepared by osmotic lysis with biotinylated proteins detected by horseradish peroxidase-linked streptavidin. I/U indicates extracts from P. falciparum infected and uninfected erythrocytes. The first four lanes in panels A, C and E contain Triton X-100 insoluble material ( Ti ) whereas the succeeding four lanes contain Triton X-100 soluble material ( Ts ). The first two lanes in panels B, D and F contain the insoluble fractions which can be pelleted by centrifugation after osmotic lysis ( Op ), the next four lanes contain the post-osmotic lysis membranous fraction ( Om ) and the final two lanes contain the osmotic lysis supernatant fraction ( Os ). The solid arrowheads highlight Triton-insoluble, infected erythrocyte-specific, trypsin-sensitive biotinylated proteins. The empty arrowhead highlights a ~110 kDa Triton-soluble, infected erythrocyte-specific, trypsin-sensitive biotinylated protein which also partitions into the osmotic lysis pellet. Panel C and D . Blots A and B respectively, were re-probed with an anti-PfEMP1 antiserum to show co-localization with a biotin-labelled, infected erythrocyte-specific, trypsin-sensitive protein expressed by the R29 parasite clone. Panel E and F . Silver stained duplicate gels of the gels used to prepare the Western blots shown in Panel A and B respectively to confirm the integrity of the protein extracts.
    Figure Legend Snippet: Detergent and osmotic lysis extracts of biotinylated P. falciparum PESAs . Panel A . Western blotting of detergent extracts from surface biotinylated P. falciparum infected erythrocytes. Biotinylated proteins were detected by horseradish peroxidase-linked streptavidin. Panel B . Western blotting of surface biotinylated P. falciparum infected erythrocyte extracts prepared by osmotic lysis with biotinylated proteins detected by horseradish peroxidase-linked streptavidin. I/U indicates extracts from P. falciparum infected and uninfected erythrocytes. The first four lanes in panels A, C and E contain Triton X-100 insoluble material ( Ti ) whereas the succeeding four lanes contain Triton X-100 soluble material ( Ts ). The first two lanes in panels B, D and F contain the insoluble fractions which can be pelleted by centrifugation after osmotic lysis ( Op ), the next four lanes contain the post-osmotic lysis membranous fraction ( Om ) and the final two lanes contain the osmotic lysis supernatant fraction ( Os ). The solid arrowheads highlight Triton-insoluble, infected erythrocyte-specific, trypsin-sensitive biotinylated proteins. The empty arrowhead highlights a ~110 kDa Triton-soluble, infected erythrocyte-specific, trypsin-sensitive biotinylated protein which also partitions into the osmotic lysis pellet. Panel C and D . Blots A and B respectively, were re-probed with an anti-PfEMP1 antiserum to show co-localization with a biotin-labelled, infected erythrocyte-specific, trypsin-sensitive protein expressed by the R29 parasite clone. Panel E and F . Silver stained duplicate gels of the gels used to prepare the Western blots shown in Panel A and B respectively to confirm the integrity of the protein extracts.

    Techniques Used: Lysis, Western Blot, Infection, Centrifugation, Staining

    9) Product Images from "Fission Yeast Asc1 Stabilizes the Interaction between Eukaryotic Initiation Factor 3a and Rps0A/uS2 for Protein Synthesis"

    Article Title: Fission Yeast Asc1 Stabilizes the Interaction between Eukaryotic Initiation Factor 3a and Rps0A/uS2 for Protein Synthesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00161-19

    The fission yeast AME complex. (A) Schematic representation of the domain structures of S. pombe (Sp) Asc1, S. cerevisiae (Sc) Arc1, and human ( Homo sapiens [Hs]) AIMP1 to AIMP3. (B) Cladogram showing the evolutionary relationship of Asc1 of S. pombe to AIMPs of humans. (C) Tandem affinity purification (TAP) tag pulldown of Asc1, ERS, and MRS proteins resolved by SDS-PAGE was visualized by silver staining. The identities of the constituent proteins (identified by MALDI-MS/MS analysis of individual bands) are indicated on the right. (D) Schematic representation of the structural organization of the fission yeast AME complex. Coimmunoprecipitation was performed with extracts prepared from strains expressing the proteins of interest in the presence or absence of Asc1. A GFP-Trap affinity resin was used to pull down the GFPs. The immunoprecipitates (IP) were then analyzed by Western immunoblotting using antibodies against GFP and HA. (E) Coimmunoprecipitation was performed with extracts prepared from ERS-HA-tagged strains expressing full-length or N-terminus-truncated Asc1-GFPs from the pREP41 plasmid. A GFP-Trap affinity resin was used to pull down the GFPs. The immunoprecipitates were then analyzed by Western immunoblotting using antibodies against GFP and HA. a.a., amino acids. (F) A GFP-Trap affinity resin was used to pull down the GFPs. The level of biotinylation was determined by the use of streptavidin conjugated to horseradish peroxidase (strep-HRP) and anti-GFP antibodies.
    Figure Legend Snippet: The fission yeast AME complex. (A) Schematic representation of the domain structures of S. pombe (Sp) Asc1, S. cerevisiae (Sc) Arc1, and human ( Homo sapiens [Hs]) AIMP1 to AIMP3. (B) Cladogram showing the evolutionary relationship of Asc1 of S. pombe to AIMPs of humans. (C) Tandem affinity purification (TAP) tag pulldown of Asc1, ERS, and MRS proteins resolved by SDS-PAGE was visualized by silver staining. The identities of the constituent proteins (identified by MALDI-MS/MS analysis of individual bands) are indicated on the right. (D) Schematic representation of the structural organization of the fission yeast AME complex. Coimmunoprecipitation was performed with extracts prepared from strains expressing the proteins of interest in the presence or absence of Asc1. A GFP-Trap affinity resin was used to pull down the GFPs. The immunoprecipitates (IP) were then analyzed by Western immunoblotting using antibodies against GFP and HA. (E) Coimmunoprecipitation was performed with extracts prepared from ERS-HA-tagged strains expressing full-length or N-terminus-truncated Asc1-GFPs from the pREP41 plasmid. A GFP-Trap affinity resin was used to pull down the GFPs. The immunoprecipitates were then analyzed by Western immunoblotting using antibodies against GFP and HA. a.a., amino acids. (F) A GFP-Trap affinity resin was used to pull down the GFPs. The level of biotinylation was determined by the use of streptavidin conjugated to horseradish peroxidase (strep-HRP) and anti-GFP antibodies.

    Techniques Used: Affinity Purification, SDS Page, Silver Staining, Tandem Mass Spectroscopy, Expressing, Western Blot, Plasmid Preparation

    10) Product Images from "Src kinase phosphorylates Notch1 to inhibit MAML binding"

    Article Title: Src kinase phosphorylates Notch1 to inhibit MAML binding

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33920-y

    Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.
    Figure Legend Snippet: Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.

    Techniques Used: Purification, Western Blot, Expressing, Dominant Negative Mutation, Sequencing, Immunoprecipitation, Over Expression, Inhibition

    11) Product Images from "Identification of Michael Acceptor-Centric Pharmacophores with Substituents That Yield Strong Thioredoxin Reductase Inhibitory Character Correlated to Antiproliferative Activity"

    Article Title: Identification of Michael Acceptor-Centric Pharmacophores with Substituents That Yield Strong Thioredoxin Reductase Inhibitory Character Correlated to Antiproliferative Activity

    Journal: Antioxidants & Redox Signaling

    doi: 10.1089/ars.2012.4909

    Irreversibility of TrxR inhibition and selenocysteine (Sec) residue targeting by the DPPen and DPPro lead analogs. (A) Oxidized recombinant rat TrxR (110 n M ) in the presence or absence of 200 μ M NADPH was incubated with different concentrations of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min, and the TrxR activity was determined by the DTNB assay. All data points are means of two independent experiments. (B) Recombinant rat TrxR (0.9 μ M ) was incubated with 20 μ M of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min in a 50 mM Tris-HCl, 1 mN EDTA, pH 7.5 buffer containing 200 μ M NADPH. The excess drug was removed by passing the protein through a NAP-5-desalting column, followed by determination of the activity of the eluted protein at indicated times using the DTNB assay. (C) Recombinant rat TrxR (0.9 μ M ) was incubated with 200 μ M NADPH and 20 μ M of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro. At different timepoints, an aliquot of enzyme mixture was withdrawn for TrxR activity measurement by the DTNB assay and N-(Biotinoyl)-N′-(Iodoacetyl) Ethylenediamine (BIAM) labeling of Sec at pH 6.5 and 8.5. Top panel: time course of TrxR enzyme activity; middle panel: horseradish peroxidase (HRP)-conjugated streptavidin detection of BIAM labeling of free selenol at pH 6.5 at various incubation times; bottom panel: HRP-conjugated streptavidin detection of BIAM labeling of free selenol and sulfhydryls at pH 8.5 at various incubation times. Results shown are representative of three independent experiments.
    Figure Legend Snippet: Irreversibility of TrxR inhibition and selenocysteine (Sec) residue targeting by the DPPen and DPPro lead analogs. (A) Oxidized recombinant rat TrxR (110 n M ) in the presence or absence of 200 μ M NADPH was incubated with different concentrations of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min, and the TrxR activity was determined by the DTNB assay. All data points are means of two independent experiments. (B) Recombinant rat TrxR (0.9 μ M ) was incubated with 20 μ M of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min in a 50 mM Tris-HCl, 1 mN EDTA, pH 7.5 buffer containing 200 μ M NADPH. The excess drug was removed by passing the protein through a NAP-5-desalting column, followed by determination of the activity of the eluted protein at indicated times using the DTNB assay. (C) Recombinant rat TrxR (0.9 μ M ) was incubated with 200 μ M NADPH and 20 μ M of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro. At different timepoints, an aliquot of enzyme mixture was withdrawn for TrxR activity measurement by the DTNB assay and N-(Biotinoyl)-N′-(Iodoacetyl) Ethylenediamine (BIAM) labeling of Sec at pH 6.5 and 8.5. Top panel: time course of TrxR enzyme activity; middle panel: horseradish peroxidase (HRP)-conjugated streptavidin detection of BIAM labeling of free selenol at pH 6.5 at various incubation times; bottom panel: HRP-conjugated streptavidin detection of BIAM labeling of free selenol and sulfhydryls at pH 8.5 at various incubation times. Results shown are representative of three independent experiments.

    Techniques Used: Inhibition, Size-exclusion Chromatography, Recombinant, Incubation, Activity Assay, DTNB Assay, Labeling

    12) Product Images from "Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6"

    Article Title: Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-11-135

    TSG-6 interferes with LPS-induced activation of NF-κB signaling. BV2 cells were stimulated with LPS in the presence and absence of TSG-6 (10 ng/ml). Cells were immunostained with a primary antibody against NF-κB p65, followed by an Alexa Fluor 594-conjugated secondary antibody. Actin filaments (green) and cell nuclei (blue) were visualized with FITC-labeled phalloidin and DAPI separately. Cell images were obtained using a confocal microscopy. (A) Typical micrographs of immunocytochemistry are shown for cytoplasmic and nuclear distribution on NF-κB p65. Scale bars, 30 μm. (B) The percentages of cells with NF-κB p65 localized to the nucleus were determined by analysis of at least 100 cells per slide. (C) Nuclear extracts were prepared and processed for chemiluminescence-based NF-κB EMSA experiments. Cells were stimulated with 100 ng/ml LPS with or without TSG-6(10 ng/ml) for the indicated periods. Nuclear extracts were incubated with a biotin-labeled NF-κB-specific oligonucleotide and further probed with streptavidin-HRP. The arrow indicates shifted DNA probe for NF-κB and free probe respectively. (D) BV2 cells were co-transfected with pNF-κB-luciferase reporter plasmid and pRL-TK control plasmid and then treated with or without LPS (100 ng/ml) in appearance or absence of rmTSG-6 (10 ng/ml) for 6 hours. NF-κB activities were measured by luciferase assay, normalized to luciferase activities of pRL-TK, and quantified as fold changes over the control (unstimulated BV2 cells). Values are mean ± SD. n = 3; ** P
    Figure Legend Snippet: TSG-6 interferes with LPS-induced activation of NF-κB signaling. BV2 cells were stimulated with LPS in the presence and absence of TSG-6 (10 ng/ml). Cells were immunostained with a primary antibody against NF-κB p65, followed by an Alexa Fluor 594-conjugated secondary antibody. Actin filaments (green) and cell nuclei (blue) were visualized with FITC-labeled phalloidin and DAPI separately. Cell images were obtained using a confocal microscopy. (A) Typical micrographs of immunocytochemistry are shown for cytoplasmic and nuclear distribution on NF-κB p65. Scale bars, 30 μm. (B) The percentages of cells with NF-κB p65 localized to the nucleus were determined by analysis of at least 100 cells per slide. (C) Nuclear extracts were prepared and processed for chemiluminescence-based NF-κB EMSA experiments. Cells were stimulated with 100 ng/ml LPS with or without TSG-6(10 ng/ml) for the indicated periods. Nuclear extracts were incubated with a biotin-labeled NF-κB-specific oligonucleotide and further probed with streptavidin-HRP. The arrow indicates shifted DNA probe for NF-κB and free probe respectively. (D) BV2 cells were co-transfected with pNF-κB-luciferase reporter plasmid and pRL-TK control plasmid and then treated with or without LPS (100 ng/ml) in appearance or absence of rmTSG-6 (10 ng/ml) for 6 hours. NF-κB activities were measured by luciferase assay, normalized to luciferase activities of pRL-TK, and quantified as fold changes over the control (unstimulated BV2 cells). Values are mean ± SD. n = 3; ** P

    Techniques Used: Activation Assay, Labeling, Confocal Microscopy, Immunocytochemistry, Incubation, Transfection, Luciferase, Plasmid Preparation

    13) Product Images from "Construction and Characterization of Moraxella catarrhalis Mutants Defective in Expression of Transferrin Receptors"

    Article Title: Construction and Characterization of Moraxella catarrhalis Mutants Defective in Expression of Transferrin Receptors

    Journal: Infection and Immunity

    doi:

    Solid-phase Tf binding assay. Equal amounts of iron-depleted whole cells from the parental and mutant Tbp derivatives were dotted onto nitrocellulose. The blots were incubated with biotinylated human holotransferrin and developed by using horseradish peroxidase-streptavidin to detect Tf binding. TbpA + B + , 7169; TbpA + B − , 7169b12; TbpA − B + , 7169 tbpA 35; TbpA − B − , 7169 tbpAB -1.
    Figure Legend Snippet: Solid-phase Tf binding assay. Equal amounts of iron-depleted whole cells from the parental and mutant Tbp derivatives were dotted onto nitrocellulose. The blots were incubated with biotinylated human holotransferrin and developed by using horseradish peroxidase-streptavidin to detect Tf binding. TbpA + B + , 7169; TbpA + B − , 7169b12; TbpA − B + , 7169 tbpA 35; TbpA − B − , 7169 tbpAB -1.

    Techniques Used: Binding Assay, Mutagenesis, Incubation

    14) Product Images from "Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation"

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000666

    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Figure Legend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Techniques Used: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.
    Figure Legend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Techniques Used: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P
    Figure Legend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Techniques Used: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P
    Figure Legend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Techniques Used: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P
    Figure Legend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Techniques Used: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    15) Product Images from "Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts"

    Article Title: Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts

    Journal: Analytical Chemistry

    doi: 10.1021/ac900281n

    Detection of carbonyl groups formed in proteins during IEF: Day 1 zebrafish embryo protein samples were resolved by 2-DE, and the IEF was performed in rehydration buffer containing 1 mM bPA in the (A) absence or (B) presence of 20 mM NaCl or (C) under priming IEF in the presence of 20 mM NaCl at a maximal voltage of 500 V for 200 voltage hours followed by in-gel dialysis and refocusing IEF in rehydration buffer containing 1 mM bPA. Streptavidin-peroxidase blot overlay was used to probe the biotin−pentylamine adducts to reduced proteins.
    Figure Legend Snippet: Detection of carbonyl groups formed in proteins during IEF: Day 1 zebrafish embryo protein samples were resolved by 2-DE, and the IEF was performed in rehydration buffer containing 1 mM bPA in the (A) absence or (B) presence of 20 mM NaCl or (C) under priming IEF in the presence of 20 mM NaCl at a maximal voltage of 500 V for 200 voltage hours followed by in-gel dialysis and refocusing IEF in rehydration buffer containing 1 mM bPA. Streptavidin-peroxidase blot overlay was used to probe the biotin−pentylamine adducts to reduced proteins.

    Techniques Used: Electrofocusing

    Detection of carbonyl groups formed in proteins during electrolysis in an electrolyzer: (A) RNase A was electrolyzed in 1% acetic acid with 0.1 M NaCl at 100 V for 4 h and derivatized with biotin-pentylamine (bPA) or biotin-LC-hydrazide (bHz) at pH 7−8. (B) Ovalbumin (OVA) was electrolyzed in 1% acetic acid with 0.1 M NaCl at 100 V for 4 h and derivatized with various amounts of bPA. (C) Both electroreduced RNase A and OVA were derivatized with 2,4-dinitrophenyl hydrazine (DNPH). Streptavidin-peroxidase blot overlay was used for detection of biotin-labeled proteins and immunoblotting of DNP-proteins. DNP: 2,4-dinitrophenyl.
    Figure Legend Snippet: Detection of carbonyl groups formed in proteins during electrolysis in an electrolyzer: (A) RNase A was electrolyzed in 1% acetic acid with 0.1 M NaCl at 100 V for 4 h and derivatized with biotin-pentylamine (bPA) or biotin-LC-hydrazide (bHz) at pH 7−8. (B) Ovalbumin (OVA) was electrolyzed in 1% acetic acid with 0.1 M NaCl at 100 V for 4 h and derivatized with various amounts of bPA. (C) Both electroreduced RNase A and OVA were derivatized with 2,4-dinitrophenyl hydrazine (DNPH). Streptavidin-peroxidase blot overlay was used for detection of biotin-labeled proteins and immunoblotting of DNP-proteins. DNP: 2,4-dinitrophenyl.

    Techniques Used: Electrolysis, Labeling

    16) Product Images from "Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection"

    Article Title: Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20081398

    Tim-3 is up-regulated on T cells in HIV-1 infection, and its expression correlates with parameters of HIV-1 disease progression. (a) PBMCs from HIV-1–infected and uninfected subjects were stained with antibodies against CD4, CD8, CD3, and a biotinylated polyclonal goat anti–Tim-3 antibody, followed by a secondary streptavidin–APC conjugate. Plots show events gated on the CD3 + population, and subsequently on the CD8 + or CD4 + populations, from a representative HIV-1–uninfected subject and a chronically HIV-1–infected subject. Biotinylated normal goat serum was used as a negative control. (b) The percentages of Tim-3 + cells within CD8 + and CD4 + T cell populations are indicated for nine HIV-1–uninfected individuals and 31 individuals from the CIRC cohort separated into three groups: acute/early HIV-1 infected ( 7 ), HIV-1–infected chronic progressors ( 16 ), and HIV-1–infected viral controller ( 8 ), using polyclonal goat anti–Tim-3 antibody. (c) The percentages of Tim-3 + cells within CD8 + and CD4 + T cell populations are indicated for 60 treatment-naive HIV-1–infected individuals from the UCSF OPTIONS cohort of primary infection and 9 HIV-1–uninfected controls using PE-conjugated monoclonal anti–Tim-3 antibody. Subjects from the CIRC cohort were defined as follows: acute/early = infected with HIV-1
    Figure Legend Snippet: Tim-3 is up-regulated on T cells in HIV-1 infection, and its expression correlates with parameters of HIV-1 disease progression. (a) PBMCs from HIV-1–infected and uninfected subjects were stained with antibodies against CD4, CD8, CD3, and a biotinylated polyclonal goat anti–Tim-3 antibody, followed by a secondary streptavidin–APC conjugate. Plots show events gated on the CD3 + population, and subsequently on the CD8 + or CD4 + populations, from a representative HIV-1–uninfected subject and a chronically HIV-1–infected subject. Biotinylated normal goat serum was used as a negative control. (b) The percentages of Tim-3 + cells within CD8 + and CD4 + T cell populations are indicated for nine HIV-1–uninfected individuals and 31 individuals from the CIRC cohort separated into three groups: acute/early HIV-1 infected ( 7 ), HIV-1–infected chronic progressors ( 16 ), and HIV-1–infected viral controller ( 8 ), using polyclonal goat anti–Tim-3 antibody. (c) The percentages of Tim-3 + cells within CD8 + and CD4 + T cell populations are indicated for 60 treatment-naive HIV-1–infected individuals from the UCSF OPTIONS cohort of primary infection and 9 HIV-1–uninfected controls using PE-conjugated monoclonal anti–Tim-3 antibody. Subjects from the CIRC cohort were defined as follows: acute/early = infected with HIV-1

    Techniques Used: Infection, Expressing, Staining, Negative Control

    17) Product Images from "A Neurostimulant para-Chloroamphetamine Inhibits the Arginylation Branch of the N-end Rule Pathway"

    Article Title: A Neurostimulant para-Chloroamphetamine Inhibits the Arginylation Branch of the N-end Rule Pathway

    Journal: Scientific Reports

    doi: 10.1038/srep06344

    PCA inhibits the type 1 and type 2 Arg/N-end rule pathways in vitro . (A) Artificial Arg/N-end rule substrates Arg-nsP4 and Phe-nsP4, which are type 1 and type 2 model substrates, respectively, were stabilized by PCA. Tripartite DHFR-Ub-X-nsP4 fusion proteins were expressed in vitro with biotin labels using rabbit reticulocyte lysates and biotinylated lysyl tRNA. Total reaction mixtures were subjected to SDS-PAGE followed by Western blot using horseradish peroxidase (HRP)-conjugated streptavidin. Cotranslational cleavage events by deubiquitinating enzymes (DUB) yields long-lived DHFR-Ub references and X-nsP4 substrates as indicated. Asterisk indicates nonspecific signals or cleaved forms of X-nsP4. The in vitro expression/degradation reactions were performed in the presence of 0, 1, 2 mM PCA for 90 min. (B) Relative amounts of remaining X-nsP4 proteins normalized by DHFR-Ub in the presence/absence of PCA. Multiple film images were quantified by densitometry. (C) Otherwise short-lived RGS4 and RGS5, physiological Arg/N-end rule substrates, were stabilized by PCA. As in Fig. 1 A except that a proteasome core particle subunit α7 was used as loading control. (D) Compounds which are structurally similar to PCA did not inhibit the Arg/N-end rule pathway. Unlike PCA, PMMA, PMA, or PMTA exhibited little effects on RGS4 level compared to control (DMSO). Cropped gels/blots are used in (C) and (D). (E) Quantification of RGS4 levels, which are normalized to those of α7. Data represent mean ± SD (n = 3).
    Figure Legend Snippet: PCA inhibits the type 1 and type 2 Arg/N-end rule pathways in vitro . (A) Artificial Arg/N-end rule substrates Arg-nsP4 and Phe-nsP4, which are type 1 and type 2 model substrates, respectively, were stabilized by PCA. Tripartite DHFR-Ub-X-nsP4 fusion proteins were expressed in vitro with biotin labels using rabbit reticulocyte lysates and biotinylated lysyl tRNA. Total reaction mixtures were subjected to SDS-PAGE followed by Western blot using horseradish peroxidase (HRP)-conjugated streptavidin. Cotranslational cleavage events by deubiquitinating enzymes (DUB) yields long-lived DHFR-Ub references and X-nsP4 substrates as indicated. Asterisk indicates nonspecific signals or cleaved forms of X-nsP4. The in vitro expression/degradation reactions were performed in the presence of 0, 1, 2 mM PCA for 90 min. (B) Relative amounts of remaining X-nsP4 proteins normalized by DHFR-Ub in the presence/absence of PCA. Multiple film images were quantified by densitometry. (C) Otherwise short-lived RGS4 and RGS5, physiological Arg/N-end rule substrates, were stabilized by PCA. As in Fig. 1 A except that a proteasome core particle subunit α7 was used as loading control. (D) Compounds which are structurally similar to PCA did not inhibit the Arg/N-end rule pathway. Unlike PCA, PMMA, PMA, or PMTA exhibited little effects on RGS4 level compared to control (DMSO). Cropped gels/blots are used in (C) and (D). (E) Quantification of RGS4 levels, which are normalized to those of α7. Data represent mean ± SD (n = 3).

    Techniques Used: In Vitro, SDS Page, Western Blot, Expressing

    18) Product Images from "Cysteine 893 is a target of regulatory thiol modifications of GluA1 AMPA receptors"

    Article Title: Cysteine 893 is a target of regulatory thiol modifications of GluA1 AMPA receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171489

    Association of SAP97 with nNOS. (A, B) Binding of nNOS to biotinylated MAGUK proteins PSD-95 (A) and SAP97 (B) shown in streptavidin pulldown assays. (C) GluA1 and nNOS co-immunoprecipitate with SAP97 in detergent extracts from postnatal day 7 rat cortex. SAP97 N-terminal domain–specific antiserum (SAP97 N ) was used for the immunoprecipitation, whereas the corresponding preimmune serum served as a control (CTL). The panel on the right shows the lysates. (D) GluA1, SAP97 and PSD-95 co-immunoprecipitate with nNOS in detergent extracts from postnatal day 7 rat cortex. Monoclonal nNOS antibody was used for specific immunoprecipitation, whereas monoclonal anti-MAP-2 served as control. Lysates are shown in the lower panel. (E) Streptavidin pulldown assay showing association of GluA1 to biotinylated nNOS. Immunoblots show representative images of experiments performed three or more times.
    Figure Legend Snippet: Association of SAP97 with nNOS. (A, B) Binding of nNOS to biotinylated MAGUK proteins PSD-95 (A) and SAP97 (B) shown in streptavidin pulldown assays. (C) GluA1 and nNOS co-immunoprecipitate with SAP97 in detergent extracts from postnatal day 7 rat cortex. SAP97 N-terminal domain–specific antiserum (SAP97 N ) was used for the immunoprecipitation, whereas the corresponding preimmune serum served as a control (CTL). The panel on the right shows the lysates. (D) GluA1, SAP97 and PSD-95 co-immunoprecipitate with nNOS in detergent extracts from postnatal day 7 rat cortex. Monoclonal nNOS antibody was used for specific immunoprecipitation, whereas monoclonal anti-MAP-2 served as control. Lysates are shown in the lower panel. (E) Streptavidin pulldown assay showing association of GluA1 to biotinylated nNOS. Immunoblots show representative images of experiments performed three or more times.

    Techniques Used: Binding Assay, Immunoprecipitation, CTL Assay, Western Blot

    19) Product Images from "Elucidating the Crucial Role of Poly N-Acetylglucosamine from Staphylococcus aureus in Cellular Adhesion and Pathogenesis"

    Article Title: Elucidating the Crucial Role of Poly N-Acetylglucosamine from Staphylococcus aureus in Cellular Adhesion and Pathogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124216

    PIA/PNAG production and adherence of S . aureus to nasal epithelial cells. (A) PIA/PNAG was extracted from S . aureus strains and detected using WGA-biotin. Following incubation with HRP-streptavidin, PIA/PNAG was visualized by chemiluminescence detection. (B) The adherence of bacteria to RPMI 2650 cells was determined using an adherence assay. The number of bacteria that adhered to the cells was determined by CFU enumeration, and the average number of bacteria adhered to each RPMI 2650 cell was calculated. S . carnosus TM300 was used as a control. Significant differences are denoted with *** p -value
    Figure Legend Snippet: PIA/PNAG production and adherence of S . aureus to nasal epithelial cells. (A) PIA/PNAG was extracted from S . aureus strains and detected using WGA-biotin. Following incubation with HRP-streptavidin, PIA/PNAG was visualized by chemiluminescence detection. (B) The adherence of bacteria to RPMI 2650 cells was determined using an adherence assay. The number of bacteria that adhered to the cells was determined by CFU enumeration, and the average number of bacteria adhered to each RPMI 2650 cell was calculated. S . carnosus TM300 was used as a control. Significant differences are denoted with *** p -value

    Techniques Used: Whole Genome Amplification, Incubation

    20) Product Images from "Involvement of Iron in Biofilm Formation by Staphylococcus aureus"

    Article Title: Involvement of Iron in Biofilm Formation by Staphylococcus aureus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034388

    Effect of iron on adherence of S. aureus to solid surfaces and PIA synthesis. S. aureus SA113 (A) and clinical strains, i.e. , SA130, SA148, SA229, SA435 (C) were inoculated in TSBg that contained 0 µM and 25 µM of PGG (TSBg-PGG) or supplemented with FeSO 4 in 9-cm petri dishes. After 24 h incubation, biofilm that formed on the plate was photographed. The plates were tilted and photographed to show the attachment of cells to the petri plate and the clumping of cells in the medium in the lower half of the plate. (B) PIA was extracted from S. aureus SA113 that had been cultured for 24 h in TSBg or TSBg-PGG medium containing 0, 50 and 100 µM FeSO 4 . PIA was detected using WGA-biotin. After incubation with HRP-streptavidin, the spots were visualized by chemiluminescence detection.
    Figure Legend Snippet: Effect of iron on adherence of S. aureus to solid surfaces and PIA synthesis. S. aureus SA113 (A) and clinical strains, i.e. , SA130, SA148, SA229, SA435 (C) were inoculated in TSBg that contained 0 µM and 25 µM of PGG (TSBg-PGG) or supplemented with FeSO 4 in 9-cm petri dishes. After 24 h incubation, biofilm that formed on the plate was photographed. The plates were tilted and photographed to show the attachment of cells to the petri plate and the clumping of cells in the medium in the lower half of the plate. (B) PIA was extracted from S. aureus SA113 that had been cultured for 24 h in TSBg or TSBg-PGG medium containing 0, 50 and 100 µM FeSO 4 . PIA was detected using WGA-biotin. After incubation with HRP-streptavidin, the spots were visualized by chemiluminescence detection.

    Techniques Used: Incubation, Cell Culture, Whole Genome Amplification

    21) Product Images from "Src kinase phosphorylates Notch1 to inhibit MAML binding"

    Article Title: Src kinase phosphorylates Notch1 to inhibit MAML binding

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33920-y

    Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.
    Figure Legend Snippet: Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.

    Techniques Used: Purification, Western Blot, Expressing, Dominant Negative Mutation, Sequencing, Immunoprecipitation, Over Expression, Inhibition

    22) Product Images from "Methylglyoxal in cells elicits a negative feedback loop entailing transglutaminase 2 and glyoxalase 1"

    Article Title: Methylglyoxal in cells elicits a negative feedback loop entailing transglutaminase 2 and glyoxalase 1

    Journal: Redox Biology

    doi: 10.1016/j.redox.2013.12.024

    Confirmation of glyoxalase 1 (GlxI) as a transglutaminase 2 (TG2) substrate. (A) HeLa cell lysate was subjected to the in vitro transamidation reaction with endogenous TG2 and substrates in the presence of 1 μg/μl bPA. The bPA labeled proteins were pulled down using streptavidin-agarose and applied to the biotin overlay assay (upper panel) and immunoblotting (lower panel). (B) Purified rhGlxI was subjected to the in vitro transamidation reaction and applied to the biotin overlay assay (upper panel) and immunoblotting (lower panel). Lane 1: TG2 only; lane 2: bPA only; lane 3–7: TG2, bPA and various reagents. CT: control, CTE: cysteamine, CTA: cystamine, MDC: monodansylcadaverine.
    Figure Legend Snippet: Confirmation of glyoxalase 1 (GlxI) as a transglutaminase 2 (TG2) substrate. (A) HeLa cell lysate was subjected to the in vitro transamidation reaction with endogenous TG2 and substrates in the presence of 1 μg/μl bPA. The bPA labeled proteins were pulled down using streptavidin-agarose and applied to the biotin overlay assay (upper panel) and immunoblotting (lower panel). (B) Purified rhGlxI was subjected to the in vitro transamidation reaction and applied to the biotin overlay assay (upper panel) and immunoblotting (lower panel). Lane 1: TG2 only; lane 2: bPA only; lane 3–7: TG2, bPA and various reagents. CT: control, CTE: cysteamine, CTA: cystamine, MDC: monodansylcadaverine.

    Techniques Used: In Vitro, Labeling, Overlay Assay, Purification

    23) Product Images from "Identification of Fibronectin-Binding Proteins in Mycoplasma gallisepticum Strain R "

    Article Title: Identification of Fibronectin-Binding Proteins in Mycoplasma gallisepticum Strain R

    Journal: Infection and Immunity

    doi: 10.1128/IAI.74.3.1777-1785.2006

    Investigation of putative transmembrane regions of PlpA. GxxxG (GG 4 ), IxxxI (II 4 ), and GxxxGxxxG (GG 4 E) motifs from PlpA were examined for their abilities to interact with membranes. Liposomes were generated in the presence of biotinylated peptides representing these motifs. Excess peptide was removed by washing. Liposomes were immobilized on paraffin wax, after which incorporated peptides were detected with streptavidin-HRP. (A) Empty liposomes, the hydrophilic peptide (PlpA − ), the GG 4 peptide, and the II 4 peptide did not show any reactivity, indicating that no peptide was incorporated during synthesis. The ponticulin peptide and the expanded motif of GG 4 did show reactivity (arrows), indicating that they were incorporated into the liposomes. In addition, biotinylated peptides were introduced into the membrane of M. gallisepticum by electroporation. Resulting cells were spotted onto nitrocellulose for the detection of embedded peptide by streptavidin-HRP. (B) Panels are labeled with the name of each peptide; those marked with a plus sign represent electroporated cells used to detect embedding, and those marked with a minus sign represent nonpulsed cells mixed with peptides to detect nonspecific binding. The hydrophilic peptide (PlpA − ), the GG 4 peptide, and the II 4 peptide did not embed in the membrane or bind to it nonspecifically. The ponticulin peptide embedded in the membrane but also bound the cell surface nonspecifically. The expanded repeat of GG 4 embedded in the cell membrane and did not bind nonspecifically.
    Figure Legend Snippet: Investigation of putative transmembrane regions of PlpA. GxxxG (GG 4 ), IxxxI (II 4 ), and GxxxGxxxG (GG 4 E) motifs from PlpA were examined for their abilities to interact with membranes. Liposomes were generated in the presence of biotinylated peptides representing these motifs. Excess peptide was removed by washing. Liposomes were immobilized on paraffin wax, after which incorporated peptides were detected with streptavidin-HRP. (A) Empty liposomes, the hydrophilic peptide (PlpA − ), the GG 4 peptide, and the II 4 peptide did not show any reactivity, indicating that no peptide was incorporated during synthesis. The ponticulin peptide and the expanded motif of GG 4 did show reactivity (arrows), indicating that they were incorporated into the liposomes. In addition, biotinylated peptides were introduced into the membrane of M. gallisepticum by electroporation. Resulting cells were spotted onto nitrocellulose for the detection of embedded peptide by streptavidin-HRP. (B) Panels are labeled with the name of each peptide; those marked with a plus sign represent electroporated cells used to detect embedding, and those marked with a minus sign represent nonpulsed cells mixed with peptides to detect nonspecific binding. The hydrophilic peptide (PlpA − ), the GG 4 peptide, and the II 4 peptide did not embed in the membrane or bind to it nonspecifically. The ponticulin peptide embedded in the membrane but also bound the cell surface nonspecifically. The expanded repeat of GG 4 embedded in the cell membrane and did not bind nonspecifically.

    Techniques Used: Generated, Electroporation, Labeling, Binding Assay

    24) Product Images from "Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity"

    Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.459

    Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), filipin III (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m
    Figure Legend Snippet: Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), filipin III (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m

    Techniques Used: Transduction, Labeling, Confocal Microscopy, Purification, Incubation

    25) Product Images from "A Repeat Sequence Causes Competition of ColE1-Type Plasmids"

    Article Title: A Repeat Sequence Causes Competition of ColE1-Type Plasmids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061668

    Exchange of RNA polymerase from DR region to RNAIp and RNAIIp . (A) Sequences of RNAIp and RNAIIp probes. (B) Purified RNA polymerase (RP) was added to 32 P-labeled-DR probe (Fig. 7A). Unlabeled RNAIIp and RNAIp probes, which contained sequences shown in (A), were used to analyze exchange of RNA polymerase from DR region to RNAIp and RNAIIp . (C) DNA-protein complexes that contained a biotinylated-DR probe (Bio-DR) and RNA polymerase (Bio-DR/RP) were captured using streptavidin-coated magnetic beads. After unbound RNA polymerase had been removed, 32 P-labeled RNAIIp and RNAIp probes were added to RNA polymerase-DR complex to analyze exchange of RNA polymerase from repeats to RNAIp and RNAIIp . Probe 167 was used as a negative control.
    Figure Legend Snippet: Exchange of RNA polymerase from DR region to RNAIp and RNAIIp . (A) Sequences of RNAIp and RNAIIp probes. (B) Purified RNA polymerase (RP) was added to 32 P-labeled-DR probe (Fig. 7A). Unlabeled RNAIIp and RNAIp probes, which contained sequences shown in (A), were used to analyze exchange of RNA polymerase from DR region to RNAIp and RNAIIp . (C) DNA-protein complexes that contained a biotinylated-DR probe (Bio-DR) and RNA polymerase (Bio-DR/RP) were captured using streptavidin-coated magnetic beads. After unbound RNA polymerase had been removed, 32 P-labeled RNAIIp and RNAIp probes were added to RNA polymerase-DR complex to analyze exchange of RNA polymerase from repeats to RNAIp and RNAIIp . Probe 167 was used as a negative control.

    Techniques Used: Purification, Labeling, Magnetic Beads, Negative Control

    Analysis of proteins that bind to 15-bp repeats. An E. coli MG1655 lysate was mixed with a biotin-labeled probe, DR-I, which contained the entire DR region (lanes 3, 6), or probe 167 (lanes 2, 5). Proteins in the cell lysate that was bound to the probes were captured using streptavidin-coated magnetic beads and analyzed by immunoblotting using antibodies against β subunit of RNA polymerase (RNAP β)) (lanes 1–3) and σ 70 (lanes 4–6). Lanes 1 and 4 were loaded with 0.05% cell lysate.
    Figure Legend Snippet: Analysis of proteins that bind to 15-bp repeats. An E. coli MG1655 lysate was mixed with a biotin-labeled probe, DR-I, which contained the entire DR region (lanes 3, 6), or probe 167 (lanes 2, 5). Proteins in the cell lysate that was bound to the probes were captured using streptavidin-coated magnetic beads and analyzed by immunoblotting using antibodies against β subunit of RNA polymerase (RNAP β)) (lanes 1–3) and σ 70 (lanes 4–6). Lanes 1 and 4 were loaded with 0.05% cell lysate.

    Techniques Used: Labeling, Magnetic Beads

    26) Product Images from "Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints"

    Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116237

    The mAbs recognize an epitope that contains an O-linked glycan modification of the octapeptide K-E/A-P-A-P-T-T-T/A/P. Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.
    Figure Legend Snippet: The mAbs recognize an epitope that contains an O-linked glycan modification of the octapeptide K-E/A-P-A-P-T-T-T/A/P. Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.

    Techniques Used: Modification, Expressing, Recombinant, SDS Page, Mutagenesis, Variant Assay, Labeling

    mAb-7h12 can measure lubricin in human plasma, serum, and synovial fluid by competition ELISA. (A) Photograph showing the result of a competition ELISA performed in triplicate. Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with purified lubricin (the concentrations of purified lubricin are indicated on top of each column) and added to lubricin-coated wells. After several washes, the mAb bound to the lubricin-coated wells, was detected colorimetrically using horseradish peroxidase (HRP) conjugated to streptavidin. Note when antibody is not pre-incubated with lubricin (0 μg/ml), all antibody binds to the pre-coated wells and the HRP-streptavidin detection of bound antibody turns these wells dark blue. In contrast, pre-incubating the antibody with increasing amounts of purified lubricin reduces antibody binding to the wells and there is less color formation by HRP-streptavidin. (B) Standard curve derived from the O.D. 415 nm values for the competition ELISA shown in panel A . The x-axis indicates the concentration of lubricin (μg/ml) that was pre-incubated with the antibody; the y-axis indicates the average difference in O.D. reading compared to the 0 μg/ml lubricin control. (C) Photograph showing the result of a competition ELISA performed in duplicate (individual rows). Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with 1:50 dilutions of human plasma or serum, or 1:50,000 dilutions of human synovial fluid. The plasma samples are from patients with CACP (CA697–1, CA698–1, CA698–2) and their unaffected family members (CA697–2, CA698–4, CA698–5). The serum samples are from healthy controls (OT701–1; OT702–1). The synovial fluid samples are from patients with OA, RA, or CACP (CA). Note that plasma and synovial fluid from patients with CACP do not reduce antibody binding to lubricin-coated wells as indicated by the dark blue color, whereas plasma, serum, or synovial fluid from unaffected individuals does reduce antibody binding to the lubricin-coated wells. Based upon the measured O.D. values for these samples (data not shown), no detectable lubricin is present in serum and synovial fluid from patients with CACP, whereas ~ 200 μg/ml is present in the RA and OA synovial fluid samples and ~ 0.2 μg/ml is present in the plasma and serum samples from unaffected relatives and controls. (D) mAbs 9g3, 5cll, 6a8 and 7h12 can be used interchangeably with recombinant human lubricin in a competition ELISA format.
    Figure Legend Snippet: mAb-7h12 can measure lubricin in human plasma, serum, and synovial fluid by competition ELISA. (A) Photograph showing the result of a competition ELISA performed in triplicate. Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with purified lubricin (the concentrations of purified lubricin are indicated on top of each column) and added to lubricin-coated wells. After several washes, the mAb bound to the lubricin-coated wells, was detected colorimetrically using horseradish peroxidase (HRP) conjugated to streptavidin. Note when antibody is not pre-incubated with lubricin (0 μg/ml), all antibody binds to the pre-coated wells and the HRP-streptavidin detection of bound antibody turns these wells dark blue. In contrast, pre-incubating the antibody with increasing amounts of purified lubricin reduces antibody binding to the wells and there is less color formation by HRP-streptavidin. (B) Standard curve derived from the O.D. 415 nm values for the competition ELISA shown in panel A . The x-axis indicates the concentration of lubricin (μg/ml) that was pre-incubated with the antibody; the y-axis indicates the average difference in O.D. reading compared to the 0 μg/ml lubricin control. (C) Photograph showing the result of a competition ELISA performed in duplicate (individual rows). Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with 1:50 dilutions of human plasma or serum, or 1:50,000 dilutions of human synovial fluid. The plasma samples are from patients with CACP (CA697–1, CA698–1, CA698–2) and their unaffected family members (CA697–2, CA698–4, CA698–5). The serum samples are from healthy controls (OT701–1; OT702–1). The synovial fluid samples are from patients with OA, RA, or CACP (CA). Note that plasma and synovial fluid from patients with CACP do not reduce antibody binding to lubricin-coated wells as indicated by the dark blue color, whereas plasma, serum, or synovial fluid from unaffected individuals does reduce antibody binding to the lubricin-coated wells. Based upon the measured O.D. values for these samples (data not shown), no detectable lubricin is present in serum and synovial fluid from patients with CACP, whereas ~ 200 μg/ml is present in the RA and OA synovial fluid samples and ~ 0.2 μg/ml is present in the plasma and serum samples from unaffected relatives and controls. (D) mAbs 9g3, 5cll, 6a8 and 7h12 can be used interchangeably with recombinant human lubricin in a competition ELISA format.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Labeling, Incubation, Purification, Binding Assay, Derivative Assay, Concentration Assay, Recombinant

    The mAbs detect an octapeptide motif present in the first mucin-like domain of human lubricin. Different domains of lubricin (depicted in the top panel beneath a schematic of the protein and its 12 coding exons) were cloned into a mammalian expression construct downstream of a signal peptide sequence and a Hemaglutinin epitope-tag (HA) sequence. Domain Mu1b was also fused to a human IgG-Fc fragment (Mu1b-Fc). An octapeptide motif (KEPAPTTT), which occurs multiple times within the first mucin-like domain (Mu1), was also fused to the IgG-Fc fragment (T-Fc). These constructs were transiently transfected into 293T cells. Serum-free conditioned media were collected and subjected to SDS-PAGE on 4–20% gradient gels. Anti-HA antibody detected all recombinant proteins in the media (HA), although weak immunodetectable bands occurred for Mu1a and Mu1c. Monoclonal antibodies 9g3, 7h12, 5c11 and 6a8 detected the secreted first mucin domain Mu1, Mu1a and Mu1c, but not Mu1b or the second mucin domain Mu2. Mu1b is the first 98 AA of the first mucin domain and does not contain KEPAPTTT; instead it has “KEPTPTT” and other “ETTT”-containing repeats. The only peptide motifs that Mu1a and Mu1c share is: “KEPAPTTP”. Antibodies 9g3, 7h12 and 6a8 also strongly recognized the KEPAPTTT containing recombinant protein (T-Fc), while 5c11 weakly interacted with T-Fc. Horseradish peroxidase (HRP) conjugated anti-mouse IgG, which was used as secondary antibody to detect 9g3, showed weak cross reactivity to human IgG-Fc. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled 7h12, 5c11 and 6a8, and showed no cross-reactivity by itself (data not shown).
    Figure Legend Snippet: The mAbs detect an octapeptide motif present in the first mucin-like domain of human lubricin. Different domains of lubricin (depicted in the top panel beneath a schematic of the protein and its 12 coding exons) were cloned into a mammalian expression construct downstream of a signal peptide sequence and a Hemaglutinin epitope-tag (HA) sequence. Domain Mu1b was also fused to a human IgG-Fc fragment (Mu1b-Fc). An octapeptide motif (KEPAPTTT), which occurs multiple times within the first mucin-like domain (Mu1), was also fused to the IgG-Fc fragment (T-Fc). These constructs were transiently transfected into 293T cells. Serum-free conditioned media were collected and subjected to SDS-PAGE on 4–20% gradient gels. Anti-HA antibody detected all recombinant proteins in the media (HA), although weak immunodetectable bands occurred for Mu1a and Mu1c. Monoclonal antibodies 9g3, 7h12, 5c11 and 6a8 detected the secreted first mucin domain Mu1, Mu1a and Mu1c, but not Mu1b or the second mucin domain Mu2. Mu1b is the first 98 AA of the first mucin domain and does not contain KEPAPTTT; instead it has “KEPTPTT” and other “ETTT”-containing repeats. The only peptide motifs that Mu1a and Mu1c share is: “KEPAPTTP”. Antibodies 9g3, 7h12 and 6a8 also strongly recognized the KEPAPTTT containing recombinant protein (T-Fc), while 5c11 weakly interacted with T-Fc. Horseradish peroxidase (HRP) conjugated anti-mouse IgG, which was used as secondary antibody to detect 9g3, showed weak cross reactivity to human IgG-Fc. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled 7h12, 5c11 and 6a8, and showed no cross-reactivity by itself (data not shown).

    Techniques Used: Clone Assay, Expressing, Construct, Sequencing, Transfection, SDS Page, Recombinant, Labeling

    Immunoprecipitation of lubricin from human serum, plasma and synovial fluid. Biotin-labeled mAb-7h12 was mixed with plasma samples from a patient with CACP (CA697–1) or his unaffected father (CA697–2), serum samples from a patient with rheumatoid arthritis (RA) or an age- and sex-matched healthy control (Control), synovial fluid samples from a patient with osteoarthritis (OA), rheumatoid arthritis (RA), or CACP (CA), and with human (hLub-IP) and bovine (bLub-IP) lubricin that had been purified from synovial fluid. Antibody was recovered using streptavidin beads and proteins co-precipitated with mAb-7h12 were eluted, subjected to SDS-PAGE, transferred to PVDF, and immunodetected with mAb-7h12 (upper panel) or polyclonal Ab-J108N (lower panel). Note mAb-7h12 immunoprecipitated lubricin from plasma and serum that is also recognized by polyclonal antibody J108N. In contrast, lubricin immunoprecipitated from OA and RA synovial fluid is not recognized by polyclonal antibody J108N. J108N also does not recognize immunoprecipitated purified human lubricin (hLub-IP). The is no non-specific binding of lubricin to streptavidin beads as demonstrated by the lack of immunodetectable protein when mAb-7h12 is not used in the co-IP (no Ab control) of plasma from the unaffected CACP parent or synovial fluid from a patient with RA. As positive controls for immunodetection purified human and bovine lubricin (hLub and bLub) were directly subjected to SDS-PAGE for immunodetection.
    Figure Legend Snippet: Immunoprecipitation of lubricin from human serum, plasma and synovial fluid. Biotin-labeled mAb-7h12 was mixed with plasma samples from a patient with CACP (CA697–1) or his unaffected father (CA697–2), serum samples from a patient with rheumatoid arthritis (RA) or an age- and sex-matched healthy control (Control), synovial fluid samples from a patient with osteoarthritis (OA), rheumatoid arthritis (RA), or CACP (CA), and with human (hLub-IP) and bovine (bLub-IP) lubricin that had been purified from synovial fluid. Antibody was recovered using streptavidin beads and proteins co-precipitated with mAb-7h12 were eluted, subjected to SDS-PAGE, transferred to PVDF, and immunodetected with mAb-7h12 (upper panel) or polyclonal Ab-J108N (lower panel). Note mAb-7h12 immunoprecipitated lubricin from plasma and serum that is also recognized by polyclonal antibody J108N. In contrast, lubricin immunoprecipitated from OA and RA synovial fluid is not recognized by polyclonal antibody J108N. J108N also does not recognize immunoprecipitated purified human lubricin (hLub-IP). The is no non-specific binding of lubricin to streptavidin beads as demonstrated by the lack of immunodetectable protein when mAb-7h12 is not used in the co-IP (no Ab control) of plasma from the unaffected CACP parent or synovial fluid from a patient with RA. As positive controls for immunodetection purified human and bovine lubricin (hLub and bLub) were directly subjected to SDS-PAGE for immunodetection.

    Techniques Used: Immunoprecipitation, Labeling, Purification, SDS Page, Binding Assay, Co-Immunoprecipitation Assay, Immunodetection

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    Article Snippet: .. Sodium cyanoborohydride, biotin hydrazide (BH), ultralinked immobilized monomeric avidin, D-biotin, protein A/G chromatography cartridges, UltraLink Biosupport™, Slide-A-Lyzer™ dialysis cassettes, biotinylated alkaline phosphatase, biotinylated horseradish peroxidase, biotinylated protein A and biotinylated protein G were purchased from Pierce (Rockford, IL, USA). .. Iodoacetamide, dithiothreitol (DTT), glycine, α-cyano-4-hydroxy-cinnamic acid (CHCA), proteomics grade N-p-tosyl-phenylalanine chloromethyl ketone (TPCK)-treated trypsin, ammonium bicarbonate, guanidine, and L-cysteine were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

    Avidin-Biotin Assay:

    Article Title: Oxidative stress induced carbonylation in human plasma
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    Article Title: Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells
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    Blocking Assay:

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    Incubation:

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    Western Blot:

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    Binding Assay:

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    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin conjugated hrp
    Photoaffinity labeling of various PrP species. <t>Streptavidin-HRP-probed</t> blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.
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    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Journal: Journal of Proteome Research

    Article Title: Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis

    doi: 10.1021/pr200918f

    Figure Lengend Snippet: Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Article Snippet: Immunoprecipitated protein was separated by SDS-PAGE and immunoblotted with either anti-FLAG M2-Peroxidase (HRP) antibody (Sigma) or Horseradish Peroxidase-Conjugated Streptavidin (Thermo Scientific).

    Techniques: Modification, Transgenic Assay, Expressing, FLAG-tag, Labeling, Protein Extraction, Generated, Affinity Purification, SDS Page, Recombinant

    Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Journal: Biochemistry

    Article Title: Prion Nucleation Site Unmasked by Transient Interaction with Phospholipid Cofactor

    doi: 10.1021/bi4014825

    Figure Lengend Snippet: Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Article Snippet: The resulting photoaffinity-labeled molecules were run on SDS-PAGE, transferred to PVDF, blocked with a 2.5% solution of bovine serum albumin (Fisher Scientific, Pittsburgh, PA), and incubated with streptavidin-conjugated HRP (ThermoFisher Scientific, Rockford, IL) at a 1:10 000 dilution before being washed with TBST and developed with SuperSignal West Femto maximum sensitivity substrate (ThermoFisher Scientific, Rockford, IL).

    Techniques: Labeling, Incubation

    TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Article Snippet: Samples were subsequently immunoprecipitated with an anti-GSK3β antibody and separated on SDS–PAGE followed by streptavidin conjugated to HRP (Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro, Real-time Polymerase Chain Reaction, Expressing