streptavidin -- horseradish Thermo Fisher Search Results


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  • 90
    Thermo Fisher streptavidin hrp
    The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. <t>Str-HRP,</t> HRP-conjugated <t>streptavidin.</t> ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher streptavidin horseradish conjugate
    The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. <t>Str-HRP,</t> HRP-conjugated <t>streptavidin.</t> ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.
    Streptavidin Horseradish Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher horseradish conjugated streptavidin
    The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. <t>Str-HRP,</t> HRP-conjugated <t>streptavidin.</t> ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.
    Horseradish Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher streptavidin horseradish
    The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. <t>Str-HRP,</t> HRP-conjugated <t>streptavidin.</t> ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.
    Streptavidin Horseradish, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher streptavidin hrp conjugate
    Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. <t>SAv-HRP,</t> <t>streptavidin–horseradish</t> peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3
    Streptavidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 626 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher horseradish peroxidase streptavidin
    Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. <t>SAv-HRP,</t> <t>streptavidin–horseradish</t> peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3
    Horseradish Peroxidase Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher streptavidin
    Amplified microscopic detection of PTHR 1 s using the ligand PTH-HRP. The primary reaction of this ligand was with biotin-phenol (“protocol B”); the secondary detection with either <t>streptavidin-HRP</t> + TrueBlue TM or streptavidin-Qdots. These schemes were applied to signals generated by PTH-HRP either in transfected HEK 293a cells or in HOS cells. Transmission and epifluorescence, original magnification: 100 × (TrueBlue™) or 1000 × (Qdots).
    Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher pierce streptavidin hrp
    Amplified microscopic detection of PTHR 1 s using the ligand PTH-HRP. The primary reaction of this ligand was with biotin-phenol (“protocol B”); the secondary detection with either <t>streptavidin-HRP</t> + TrueBlue TM or streptavidin-Qdots. These schemes were applied to signals generated by PTH-HRP either in transfected HEK 293a cells or in HOS cells. Transmission and epifluorescence, original magnification: 100 × (TrueBlue™) or 1000 × (Qdots).
    Pierce Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher streptavidin conjugated anti horseradish peroxidase anti hrp antibody
    Amplified microscopic detection of PTHR 1 s using the ligand PTH-HRP. The primary reaction of this ligand was with biotin-phenol (“protocol B”); the secondary detection with either <t>streptavidin-HRP</t> + TrueBlue TM or streptavidin-Qdots. These schemes were applied to signals generated by PTH-HRP either in transfected HEK 293a cells or in HOS cells. Transmission and epifluorescence, original magnification: 100 × (TrueBlue™) or 1000 × (Qdots).
    Streptavidin Conjugated Anti Horseradish Peroxidase Anti Hrp Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher streptavidin poly hrp
    Schematic of an amplification strategy using <t>streptavidin</t> <t>poly-HRP.</t> The streptavidin poly-HRP is attached to the biotinylated anti-human IL-6 detection antibody.
    Streptavidin Poly Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher hrp conjugated streptavidin
    Characterization of ZASP-skeletal muscle α-actin 1 (ACTA1) interaction in mammalian cells and in vitro with purified proteins. A , co-IP ( IP ) assay showing the interaction between the ZASP isoforms and ACTA1 in wild-type mouse vastus muscle lysates. B and C , co-IP assays showing the interaction between the indicated ZASP isoforms and ACTA1 in non-muscle (COS7/HEK293) cells. The proteins were detected with anti-FLAG and anti-HA antibodies. D , purified GST-tagged ZASP-LΔex10 (WT, A165V, and A147T), but not GST alone, binds to biotinylated G-actin ( G-actin ) in a slot blot overlay assay. Biotin-G-actin was detected with <t>HRP-streptavidin.</t> GST and GST-ZASP were detected by immunoblotting with GST antibody ( anti-GST ). Arp2/3 and BSA served as positive and negative controls for G-actin binding. E , high-speed cosedimentation assay of purified GST or the indicated GST-ZASP-LΔex10 proteins and F-actin. Representative colloidal blue-stained gel images demonstrate a shift of the indicated GST-ZASP proteins from the supernatant ( S ) to the pellet ( P ) fraction in the presence of F-actin, whereas GST remained in the supernatant. Nonlinear regression analysis of the ZASP-actin interaction is shown in the right panel (triplicate assays, data represent mean ± S.D.).
    Hrp Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher peroxidase streptavidin
    Characterization of ZASP-skeletal muscle α-actin 1 (ACTA1) interaction in mammalian cells and in vitro with purified proteins. A , co-IP ( IP ) assay showing the interaction between the ZASP isoforms and ACTA1 in wild-type mouse vastus muscle lysates. B and C , co-IP assays showing the interaction between the indicated ZASP isoforms and ACTA1 in non-muscle (COS7/HEK293) cells. The proteins were detected with anti-FLAG and anti-HA antibodies. D , purified GST-tagged ZASP-LΔex10 (WT, A165V, and A147T), but not GST alone, binds to biotinylated G-actin ( G-actin ) in a slot blot overlay assay. Biotin-G-actin was detected with <t>HRP-streptavidin.</t> GST and GST-ZASP were detected by immunoblotting with GST antibody ( anti-GST ). Arp2/3 and BSA served as positive and negative controls for G-actin binding. E , high-speed cosedimentation assay of purified GST or the indicated GST-ZASP-LΔex10 proteins and F-actin. Representative colloidal blue-stained gel images demonstrate a shift of the indicated GST-ZASP proteins from the supernatant ( S ) to the pellet ( P ) fraction in the presence of F-actin, whereas GST remained in the supernatant. Nonlinear regression analysis of the ZASP-actin interaction is shown in the right panel (triplicate assays, data represent mean ± S.D.).
    Peroxidase Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher anti streptavidin hrp antibody
    Characterization of ZASP-skeletal muscle α-actin 1 (ACTA1) interaction in mammalian cells and in vitro with purified proteins. A , co-IP ( IP ) assay showing the interaction between the ZASP isoforms and ACTA1 in wild-type mouse vastus muscle lysates. B and C , co-IP assays showing the interaction between the indicated ZASP isoforms and ACTA1 in non-muscle (COS7/HEK293) cells. The proteins were detected with anti-FLAG and anti-HA antibodies. D , purified GST-tagged ZASP-LΔex10 (WT, A165V, and A147T), but not GST alone, binds to biotinylated G-actin ( G-actin ) in a slot blot overlay assay. Biotin-G-actin was detected with <t>HRP-streptavidin.</t> GST and GST-ZASP were detected by immunoblotting with GST antibody ( anti-GST ). Arp2/3 and BSA served as positive and negative controls for G-actin binding. E , high-speed cosedimentation assay of purified GST or the indicated GST-ZASP-LΔex10 proteins and F-actin. Representative colloidal blue-stained gel images demonstrate a shift of the indicated GST-ZASP proteins from the supernatant ( S ) to the pellet ( P ) fraction in the presence of F-actin, whereas GST remained in the supernatant. Nonlinear regression analysis of the ZASP-actin interaction is shown in the right panel (triplicate assays, data represent mean ± S.D.).
    Anti Streptavidin Hrp Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher horseradish peroxidase streptavidin conjugate
    Characterization of ZASP-skeletal muscle α-actin 1 (ACTA1) interaction in mammalian cells and in vitro with purified proteins. A , co-IP ( IP ) assay showing the interaction between the ZASP isoforms and ACTA1 in wild-type mouse vastus muscle lysates. B and C , co-IP assays showing the interaction between the indicated ZASP isoforms and ACTA1 in non-muscle (COS7/HEK293) cells. The proteins were detected with anti-FLAG and anti-HA antibodies. D , purified GST-tagged ZASP-LΔex10 (WT, A165V, and A147T), but not GST alone, binds to biotinylated G-actin ( G-actin ) in a slot blot overlay assay. Biotin-G-actin was detected with <t>HRP-streptavidin.</t> GST and GST-ZASP were detected by immunoblotting with GST antibody ( anti-GST ). Arp2/3 and BSA served as positive and negative controls for G-actin binding. E , high-speed cosedimentation assay of purified GST or the indicated GST-ZASP-LΔex10 proteins and F-actin. Representative colloidal blue-stained gel images demonstrate a shift of the indicated GST-ZASP proteins from the supernatant ( S ) to the pellet ( P ) fraction in the presence of F-actin, whereas GST remained in the supernatant. Nonlinear regression analysis of the ZASP-actin interaction is shown in the right panel (triplicate assays, data represent mean ± S.D.).
    Horseradish Peroxidase Streptavidin Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher peroxidase streptavidin conjugate
    Characterization of ZASP-skeletal muscle α-actin 1 (ACTA1) interaction in mammalian cells and in vitro with purified proteins. A , co-IP ( IP ) assay showing the interaction between the ZASP isoforms and ACTA1 in wild-type mouse vastus muscle lysates. B and C , co-IP assays showing the interaction between the indicated ZASP isoforms and ACTA1 in non-muscle (COS7/HEK293) cells. The proteins were detected with anti-FLAG and anti-HA antibodies. D , purified GST-tagged ZASP-LΔex10 (WT, A165V, and A147T), but not GST alone, binds to biotinylated G-actin ( G-actin ) in a slot blot overlay assay. Biotin-G-actin was detected with <t>HRP-streptavidin.</t> GST and GST-ZASP were detected by immunoblotting with GST antibody ( anti-GST ). Arp2/3 and BSA served as positive and negative controls for G-actin binding. E , high-speed cosedimentation assay of purified GST or the indicated GST-ZASP-LΔex10 proteins and F-actin. Representative colloidal blue-stained gel images demonstrate a shift of the indicated GST-ZASP proteins from the supernatant ( S ) to the pellet ( P ) fraction in the presence of F-actin, whereas GST remained in the supernatant. Nonlinear regression analysis of the ZASP-actin interaction is shown in the right panel (triplicate assays, data represent mean ± S.D.).
    Peroxidase Streptavidin Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher peroxidase streptavidin complex
    Characterization of ZASP-skeletal muscle α-actin 1 (ACTA1) interaction in mammalian cells and in vitro with purified proteins. A , co-IP ( IP ) assay showing the interaction between the ZASP isoforms and ACTA1 in wild-type mouse vastus muscle lysates. B and C , co-IP assays showing the interaction between the indicated ZASP isoforms and ACTA1 in non-muscle (COS7/HEK293) cells. The proteins were detected with anti-FLAG and anti-HA antibodies. D , purified GST-tagged ZASP-LΔex10 (WT, A165V, and A147T), but not GST alone, binds to biotinylated G-actin ( G-actin ) in a slot blot overlay assay. Biotin-G-actin was detected with <t>HRP-streptavidin.</t> GST and GST-ZASP were detected by immunoblotting with GST antibody ( anti-GST ). Arp2/3 and BSA served as positive and negative controls for G-actin binding. E , high-speed cosedimentation assay of purified GST or the indicated GST-ZASP-LΔex10 proteins and F-actin. Representative colloidal blue-stained gel images demonstrate a shift of the indicated GST-ZASP proteins from the supernatant ( S ) to the pellet ( P ) fraction in the presence of F-actin, whereas GST remained in the supernatant. Nonlinear regression analysis of the ZASP-actin interaction is shown in the right panel (triplicate assays, data represent mean ± S.D.).
    Peroxidase Streptavidin Complex, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher streptavidin hrp reagent
    Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific <t>streptavidin</t> binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase <t>(HRP),</t> streptavidin (SA), primary antibody (1° Ab)
    Streptavidin Hrp Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Thermo Fisher streptavidin hrp complex
    Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific <t>streptavidin</t> binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase <t>(HRP),</t> streptavidin (SA), primary antibody (1° Ab)
    Streptavidin Hrp Complex, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher hrp streptavidin reaction
    Aging-associated change on ER expression and DNA methylation. ( A ) mRNA expression for ERα and ERβ in mice aorta normalized to the 18S expression. ( B ) Image on top shows a representative experiment of biotin-dCTP incorporation to DNA following no digestion (Mock) and digestion with MspI (no methylation sensitive) or HpaII (methylation sensitive).DNA was spotted in a nylon membrane and visualized with <t>streptavidin-HRP</t> method (data shown in triplicate). Bar graphs show (left) the results of densitometric analyses from pooled data for global DNA methylation and (right) percentage of specific DNA methylation at 5′ flanking region of the gene encoding ERα. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p
    Hrp Streptavidin Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher immunopure streptavidin
    Aging-associated change on ER expression and DNA methylation. ( A ) mRNA expression for ERα and ERβ in mice aorta normalized to the 18S expression. ( B ) Image on top shows a representative experiment of biotin-dCTP incorporation to DNA following no digestion (Mock) and digestion with MspI (no methylation sensitive) or HpaII (methylation sensitive).DNA was spotted in a nylon membrane and visualized with <t>streptavidin-HRP</t> method (data shown in triplicate). Bar graphs show (left) the results of densitometric analyses from pooled data for global DNA methylation and (right) percentage of specific DNA methylation at 5′ flanking region of the gene encoding ERα. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p
    Immunopure Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin poly
    Aging-associated change on ER expression and DNA methylation. ( A ) mRNA expression for ERα and ERβ in mice aorta normalized to the 18S expression. ( B ) Image on top shows a representative experiment of biotin-dCTP incorporation to DNA following no digestion (Mock) and digestion with MspI (no methylation sensitive) or HpaII (methylation sensitive).DNA was spotted in a nylon membrane and visualized with <t>streptavidin-HRP</t> method (data shown in triplicate). Bar graphs show (left) the results of densitometric analyses from pooled data for global DNA methylation and (right) percentage of specific DNA methylation at 5′ flanking region of the gene encoding ERα. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p
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    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: <t>streptavidin</t> 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).
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    Thermo Fisher streptavidin labeled horseradish peroxidase
    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: <t>streptavidin</t> 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).
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    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: <t>streptavidin</t> 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).
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    Thermo Fisher horseradish peroxidase hrp streptavidin
    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: <t>streptavidin</t> 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).
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    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: <t>streptavidin</t> 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).
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    Thermo Fisher peroxidase conjugated streptavidin
    Parp-1 binds to the BRCA2 promoter. A , MCF-7 cell nuclear extracts were mixed with a biotinylated WT probe and a mutant probe ( M ). The DNA-protein complex was isolated with <t>streptavidin-labeled</t> magnetic beads. The magnetic bead column was washed and eluates were collected. Eluted fractions were separated on 10% acrylamide gels and visualized by silver staining. The 120-kDa protein band indicates a prominent DNA-protein complex. BSA was used as a control, and its position is indicated by a black arrow . The experiment was carried out three times. E1, E2, E3 (WT probe eluates); M , mutant probe eluate. B , mass spectrometry of the ∼120-kDa protein. C , EMSAs and antibody super-shifts were performed in MCF-7 nuclear extracts using WT, mutant probes, and Parp-1 antibody. D , EMSAs were carried out in MCF-7 nuclear extracts using the end-streptavidin blocked-biotin-labeled WT and mutant probes. E , luciferase activity of Del-9 in MCF-7 cells treated with 3-AB/NU1025, two Parp-1 inhibitors, or DMSO for 12 h.
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    Parp-1 binds to the BRCA2 promoter. A , MCF-7 cell nuclear extracts were mixed with a biotinylated WT probe and a mutant probe ( M ). The DNA-protein complex was isolated with <t>streptavidin-labeled</t> magnetic beads. The magnetic bead column was washed and eluates were collected. Eluted fractions were separated on 10% acrylamide gels and visualized by silver staining. The 120-kDa protein band indicates a prominent DNA-protein complex. BSA was used as a control, and its position is indicated by a black arrow . The experiment was carried out three times. E1, E2, E3 (WT probe eluates); M , mutant probe eluate. B , mass spectrometry of the ∼120-kDa protein. C , EMSAs and antibody super-shifts were performed in MCF-7 nuclear extracts using WT, mutant probes, and Parp-1 antibody. D , EMSAs were carried out in MCF-7 nuclear extracts using the end-streptavidin blocked-biotin-labeled WT and mutant probes. E , luciferase activity of Del-9 in MCF-7 cells treated with 3-AB/NU1025, two Parp-1 inhibitors, or DMSO for 12 h.
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    Image Search Results


    The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. Str-HRP, HRP-conjugated streptavidin. ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.

    Journal: Oncotarget

    Article Title: Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes

    doi: 10.18632/oncotarget.10454

    Figure Lengend Snippet: The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. Str-HRP, HRP-conjugated streptavidin. ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.

    Article Snippet: To detect biotinylated peptides, the membranes were incubated with HRP-conjugated streptavidin in TTBS (1:40000, Ref. 434323, Life Technologies) and then developed with a chemiluminescent substrate.

    Techniques: Incubation, Binding Assay, Avidin-Biotin Assay, Western Blot, Fluorescence, Microscopy

    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after streptavidin labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with streptavidin-HRP (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P

    Journal: Molecular and Cellular Biology

    Article Title: CD9 Regulates Major Histocompatibility Complex Class II Trafficking in Monocyte-Derived Dendritic Cells

    doi: 10.1128/MCB.00202-17

    Figure Lengend Snippet: CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after streptavidin labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with streptavidin-HRP (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P

    Article Snippet: For internalization and recycling assays, biotinylated proteins were revealed using a Fujifilm LAS-4000 system after membrane incubation with streptavidin-HRP (Invitrogen).

    Techniques: Incubation, Expressing, Flow Cytometry, Cytometry, Labeling, Cell Surface Biotinylation Assay, Immunoprecipitation

    Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with streptavidin beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using streptavidin-HRP (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.

    Journal: Genes & Development

    Article Title: A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation

    doi: 10.1101/gad.305201.117

    Figure Lengend Snippet: Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with streptavidin beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using streptavidin-HRP (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.

    Article Snippet: As a loading control, 0.25 µL of the eluted proteins was spotted onto a nitrocellulose membrane and detected using streptavidin-HRP (Thermo Scientific, no. 21130).

    Techniques: Recombinant, Construct, Binding Assay, Protein Purification, Staining, SDS Page, In Vitro, Immunoprecipitation, Incubation, Mass Spectrometry, Modification, Dot Blot, Isolation

    Molecular mechanism of fursultiamine as a hepcidin antagonist. (A) Fursultiamine tightly associates with Fpn. Cells were induced to express Fpn-GFP and were pretreated with solvent (bars 1 and 2) or 10 µ M fursultiamine (bars 3–6) for 1 hour. Cells were then either not washed (cotreatment [Co-T]; black bar) or were washed with PBS 3 times (pretreatment [Pre-T]; gray bar), and 1 µ g/ml hepcidin was added for an additional 1 or 2 hours. The amount of Fpn-GFP was quantified using flow cytometry. Each bar represents at least 3 replicates, and error bars represent the standard deviation. Results were expressed as normalized amount of Fpn-GFP where hepcidin-untreated sample = 100% and hepcidin-treated (1 or 2 hours) sample = 0%. (B) Hepcidin binding to Fpn is attenuated in the presence of fursultiamine. Cells were induced to express Fpn-GFP and were pretreated with 0 or 10 µ M fursultiamine (Furs) for 30 minutes. A range of biotinylated hepcidin (B-hep) concentrations (0, 2.5, 5, and 10 µ g/ml) was added to the cells for another 30 minutes. Protein lysates were immunoprecipitated with anti-GFP Ab (ab290), and biotinylated hepcidin bound to Fpn-GFP was detected with streptavidin-HRP. The amount of immunoprecipitated Fpn-GFP was confirmed with anti-Fpn Ab. (C) Fursultiamine blocks Fpn residue C326. Cells expressing Fpn-GFP were treated with either hepcidin (0.5 and 1 µ g/ml) or fursultiamine (1, 3, 10, and 30 µ M) for 30 minutes. Cell surface Fpn-GFP was labeled with maleimide-biotinylation reagent for 30 minutes in 4C. Total protein lysates were immunoprecipitated with anti-GFP Ab (ab290), and samples were analyzed by Western blotting using strepavidin-HRP (top panel) or anti-GFP antibody (bottom panel).

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.112.083428

    Figure Lengend Snippet: Molecular mechanism of fursultiamine as a hepcidin antagonist. (A) Fursultiamine tightly associates with Fpn. Cells were induced to express Fpn-GFP and were pretreated with solvent (bars 1 and 2) or 10 µ M fursultiamine (bars 3–6) for 1 hour. Cells were then either not washed (cotreatment [Co-T]; black bar) or were washed with PBS 3 times (pretreatment [Pre-T]; gray bar), and 1 µ g/ml hepcidin was added for an additional 1 or 2 hours. The amount of Fpn-GFP was quantified using flow cytometry. Each bar represents at least 3 replicates, and error bars represent the standard deviation. Results were expressed as normalized amount of Fpn-GFP where hepcidin-untreated sample = 100% and hepcidin-treated (1 or 2 hours) sample = 0%. (B) Hepcidin binding to Fpn is attenuated in the presence of fursultiamine. Cells were induced to express Fpn-GFP and were pretreated with 0 or 10 µ M fursultiamine (Furs) for 30 minutes. A range of biotinylated hepcidin (B-hep) concentrations (0, 2.5, 5, and 10 µ g/ml) was added to the cells for another 30 minutes. Protein lysates were immunoprecipitated with anti-GFP Ab (ab290), and biotinylated hepcidin bound to Fpn-GFP was detected with streptavidin-HRP. The amount of immunoprecipitated Fpn-GFP was confirmed with anti-Fpn Ab. (C) Fursultiamine blocks Fpn residue C326. Cells expressing Fpn-GFP were treated with either hepcidin (0.5 and 1 µ g/ml) or fursultiamine (1, 3, 10, and 30 µ M) for 30 minutes. Cell surface Fpn-GFP was labeled with maleimide-biotinylation reagent for 30 minutes in 4C. Total protein lysates were immunoprecipitated with anti-GFP Ab (ab290), and samples were analyzed by Western blotting using strepavidin-HRP (top panel) or anti-GFP antibody (bottom panel).

    Article Snippet: We then used streptavidin–horseradish peroxide (Pierce) to detect biotin-hepcidin.

    Techniques: Flow Cytometry, Cytometry, Standard Deviation, Binding Assay, Immunoprecipitation, Expressing, Labeling, Western Blot

    BioID identifies RAS interactomes. a Immunoblot detection of myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms (top) and biotinylated proteins with HRP-streptavidin after affinity capture (AC) with streptavidin-conjugated beads (middle) in the absence (−) and presence (+) of biotin. Empty vector-transduced cells (bottom) serve as a negative control. n = 2. b Representative images indicated myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms, as visualized by fluorescence microscopy. Scale bar: 5 μM. n = 1. c Immunoblot detection of total (T-) and phosphorylated (P-) ERK and AKT and d Crystal Violet staining of cells stably expressing myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms. n = 2. e Hierarchical clustering of the z -scores derived from degree that the 477 interactome proteins were labeled by the indicated myc-tagged BirA*-RAS G12V proteins in triplicate cultures. f Venn diagram of the number of interactome proteins identified by BioID by the indicated BirA*-RAS G12V proteins. Where indicated, β-actin serves as a loading control

    Journal: Nature Communications

    Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability

    doi: 10.1038/s41467-018-05692-6

    Figure Lengend Snippet: BioID identifies RAS interactomes. a Immunoblot detection of myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms (top) and biotinylated proteins with HRP-streptavidin after affinity capture (AC) with streptavidin-conjugated beads (middle) in the absence (−) and presence (+) of biotin. Empty vector-transduced cells (bottom) serve as a negative control. n = 2. b Representative images indicated myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms, as visualized by fluorescence microscopy. Scale bar: 5 μM. n = 1. c Immunoblot detection of total (T-) and phosphorylated (P-) ERK and AKT and d Crystal Violet staining of cells stably expressing myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms. n = 2. e Hierarchical clustering of the z -scores derived from degree that the 477 interactome proteins were labeled by the indicated myc-tagged BirA*-RAS G12V proteins in triplicate cultures. f Venn diagram of the number of interactome proteins identified by BioID by the indicated BirA*-RAS G12V proteins. Where indicated, β-actin serves as a loading control

    Article Snippet: The same lysates were processed as described above to affinity capture biotinylated proteins, resolved, and immunoblotted with HRP-Streptavidin (Thermo Fisher Scientific, #22130; 1:10,000).

    Techniques: Plasmid Preparation, Negative Control, Fluorescence, Microscopy, Staining, Stable Transfection, Expressing, Derivative Assay, Labeling

    Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).

    Journal: Molecular Biology of the Cell

    Article Title: Dynamics of human telomerase recruitment depend on template-telomere base pairing

    doi: 10.1091/mbc.E17-11-0637

    Figure Lengend Snippet: Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).

    Article Snippet: The HaloTag modified with biotin was detected using Strepavidin-HRP (Pierce; 1:2000).

    Techniques: Binding Assay, Activity Assay, Modification, Labeling, Western Blot, Fluorescence, Imaging, Incubation, Concentration Assay, Telomerase Assay

    Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with HRP-conjugated streptavidin, and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.

    Journal: Scientific Reports

    Article Title: Simultaneous detection of nucleotide excision repair events and apoptosis-induced DNA fragmentation in genotoxin-treated cells

    doi: 10.1038/s41598-018-20527-6

    Figure Lengend Snippet: Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with HRP-conjugated streptavidin, and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.

    Article Snippet: The membrane was incubated with 1X PBS containing 2% SDS for 30 min and incubated with streptavidin-HRP conjugates (Thermo Scientific) in 1× PBS containing 2% SDS for 30 min.

    Techniques: Irradiation, Incubation, Molecular Weight, Labeling, Nucleic Acid Electrophoresis, SDS Page, Mutagenesis

    FKBP25 associates with ribosome biogenesis factors and other proteins in an RNA-dependent manner. ( A ) Schematic of BioID-based identification of cellular proteins proximal to FKBP25. ( B ) Streptavidin-HRP western blot of whole cell extracts and streptavidin capture from U2OS cells stably expressing an FKBP25 biotin ligase fusion or biotin ligase control, incubated for 24 hours in media containing 50 μM biotin. ( C ) Mass spectrometry identification of biotinylated proteins enriched in FKBP25-BirA streptavidin purifications relative to BirA control. The number of significant peptides identified relative to fold change is shown. ( D ) Enriched gene ontologies by molecular function. ( E ) FKBP25 3xFLAG-tagged co-immunoprecipitated material with and without pre-treatment with RNaseA analyzed by SDS-PAGE and visualized by silver stain. Empty vector cell lines are shown as a control. ( F ) Mass spectrometry analysis of proteins enriched in the FKBP25-FLAG sample relative to control, for samples either untreated or pre-treated with RNaseA. ( G ) Summarized gene ontology analysis by biological process. ( H ) Overlap in identified proteins between the BioID and FLAG co-immunoprecipitation experiments.

    Journal: Nucleic Acids Research

    Article Title: The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

    doi: 10.1093/nar/gkx852

    Figure Lengend Snippet: FKBP25 associates with ribosome biogenesis factors and other proteins in an RNA-dependent manner. ( A ) Schematic of BioID-based identification of cellular proteins proximal to FKBP25. ( B ) Streptavidin-HRP western blot of whole cell extracts and streptavidin capture from U2OS cells stably expressing an FKBP25 biotin ligase fusion or biotin ligase control, incubated for 24 hours in media containing 50 μM biotin. ( C ) Mass spectrometry identification of biotinylated proteins enriched in FKBP25-BirA streptavidin purifications relative to BirA control. The number of significant peptides identified relative to fold change is shown. ( D ) Enriched gene ontologies by molecular function. ( E ) FKBP25 3xFLAG-tagged co-immunoprecipitated material with and without pre-treatment with RNaseA analyzed by SDS-PAGE and visualized by silver stain. Empty vector cell lines are shown as a control. ( F ) Mass spectrometry analysis of proteins enriched in the FKBP25-FLAG sample relative to control, for samples either untreated or pre-treated with RNaseA. ( G ) Summarized gene ontology analysis by biological process. ( H ) Overlap in identified proteins between the BioID and FLAG co-immunoprecipitation experiments.

    Article Snippet: Clones were screened by western blotting with streptavidin–horse radish peroxidase (Thermo Fisher).

    Techniques: Western Blot, Stable Transfection, Expressing, Incubation, Mass Spectrometry, Immunoprecipitation, SDS Page, Silver Staining, Plasmid Preparation

    Binding between the lectin from Aleuria aurantia (AAL) and HbA 1c or Hb. The denatured HbA 1c ( filled circle ) or Hb ( open square ) were immobilized on the surface of the plate and the amount of biotinylated AAL bound to the protein was assayed by the activity of the specifically bound streptavidin-HRP

    Journal: AMB Express

    Article Title: Assay of hemoglobin A1c using lectin from Aleuria aurantia

    doi: 10.1186/s13568-016-0288-7

    Figure Lengend Snippet: Binding between the lectin from Aleuria aurantia (AAL) and HbA 1c or Hb. The denatured HbA 1c ( filled circle ) or Hb ( open square ) were immobilized on the surface of the plate and the amount of biotinylated AAL bound to the protein was assayed by the activity of the specifically bound streptavidin-HRP

    Article Snippet: After washing with PBS-T, 25 μl of high sensitivity streptavidin-HRP (1 μg/ml, Thermo Fisher Scientific, Waltham, MA, USA) in PBS with 1 mg/ml BSA was added and incubated for 1 h. After washing with PBS-T, color was developed with TMB peroxidase substrate system (KPL, Gaithersburg, MD, USA) according to the manufacturer’s instructions.

    Techniques: Binding Assay, Activity Assay

    Screening of hemoglobin A 1c -binding lectins. The denatured hemoglobin A 1c ( white ) or hemoglobin ( black ) was immobilized on the surface of the plate before the neutralization. The binding of biotinylated lectins was assayed by the activity of the specifically bound streptavidin-HRP

    Journal: AMB Express

    Article Title: Assay of hemoglobin A1c using lectin from Aleuria aurantia

    doi: 10.1186/s13568-016-0288-7

    Figure Lengend Snippet: Screening of hemoglobin A 1c -binding lectins. The denatured hemoglobin A 1c ( white ) or hemoglobin ( black ) was immobilized on the surface of the plate before the neutralization. The binding of biotinylated lectins was assayed by the activity of the specifically bound streptavidin-HRP

    Article Snippet: After washing with PBS-T, 25 μl of high sensitivity streptavidin-HRP (1 μg/ml, Thermo Fisher Scientific, Waltham, MA, USA) in PBS with 1 mg/ml BSA was added and incubated for 1 h. After washing with PBS-T, color was developed with TMB peroxidase substrate system (KPL, Gaithersburg, MD, USA) according to the manufacturer’s instructions.

    Techniques: Binding Assay, Neutralization, Activity Assay

    DEP increased the permeability of endothelial cells (A) HAEC cells were grown to confluence in transwell to form an endothelial monolayer. The cells were then treated with or without 50ug/ml DEP1 or DEP2 for 4 hours in the presence of 1ug/ml of Streptavidin-HRP. HRP activity from the bottom well was measured as the permeability of endothelial cells. (* vs. control, n=3, p

    Journal: Toxicology letters

    Article Title: Diesel exhaust particles modulate vascular endothelial cell permeability: Implication of ZO-1 Expression

    doi: 10.1016/j.toxlet.2010.05.017

    Figure Lengend Snippet: DEP increased the permeability of endothelial cells (A) HAEC cells were grown to confluence in transwell to form an endothelial monolayer. The cells were then treated with or without 50ug/ml DEP1 or DEP2 for 4 hours in the presence of 1ug/ml of Streptavidin-HRP. HRP activity from the bottom well was measured as the permeability of endothelial cells. (* vs. control, n=3, p

    Article Snippet: Cells were then rinsed with treatment media (M199/0.1%FBS) and treated with or without DEP in treatment media in the presence of 1µg/ml of Horse Radish Peroxidase-Streptavidin (HRP-Streptavidin, from Pierce Inc) in the top transwell.

    Techniques: Permeability, Activity Assay

    Anti-CoRBS and anti-cluster A Abs must engage with the same gp120 subunit in order to interact with dimeric FcγRIIIa. Indirect ELISA was performed using recombinant YU2 ΔV1V2V3V5 gp120 protein (WT), its mutated counterpart (W69A or R419D), or an equimolar mixture of both mutated gp120 proteins (W69A and R419D). gp120-coated wells were incubated with a total concentration of 1 µg/ml of primary antibodies. Antibody binding to gp120 was detected using (A) biotin-tagged dimeric rsFcγRIIIa (0.1 µg/ml) followed by the addition of HRP-conjugated streptavidin or using (B) HRP-conjugated anti-human secondary antibody. Graphs represent relative light units (R.L.U.) determined from 5 independent experiments done in quadruplicate, with the error bars indicating means ± SEM. Statistical significance was tested using an unpaired t test or a Mann-Whitney U test based on statistical normality (*, P

    Journal: Journal of Virology

    Article Title: Two Families of Env Antibodies Efficiently Engage Fc-Gamma Receptors and Eliminate HIV-1-Infected Cells

    doi: 10.1128/JVI.01823-18

    Figure Lengend Snippet: Anti-CoRBS and anti-cluster A Abs must engage with the same gp120 subunit in order to interact with dimeric FcγRIIIa. Indirect ELISA was performed using recombinant YU2 ΔV1V2V3V5 gp120 protein (WT), its mutated counterpart (W69A or R419D), or an equimolar mixture of both mutated gp120 proteins (W69A and R419D). gp120-coated wells were incubated with a total concentration of 1 µg/ml of primary antibodies. Antibody binding to gp120 was detected using (A) biotin-tagged dimeric rsFcγRIIIa (0.1 µg/ml) followed by the addition of HRP-conjugated streptavidin or using (B) HRP-conjugated anti-human secondary antibody. Graphs represent relative light units (R.L.U.) determined from 5 independent experiments done in quadruplicate, with the error bars indicating means ± SEM. Statistical significance was tested using an unpaired t test or a Mann-Whitney U test based on statistical normality (*, P

    Article Snippet: Horseradish peroxidase (HRP)-conjugated antibody specific for the Fc region of human IgG (Pierce) or high-sensitivity streptavidin-HRP (Thermo Fisher) (1/1,000 in blocking buffer) was added for 1 h at room temperature, followed by four washes.

    Techniques: Indirect ELISA, Recombinant, Incubation, Concentration Assay, Binding Assay, MANN-WHITNEY

    Dimeric FcγRIIIa engagement by HIV + sera is driven by anti-CoRBS and anti-cluster A Abs. (A) Indirect ELISA using recombinant YU2 ΔV1V2V3V5 gp120 protein (0.25 µg/ml). gp120-coated wells were incubated with sera from 15 chronically HIV-1-infected individuals with or without A32 Fab and/or 17b Fab fragments (1 µg/ml). (B) Indirect ELISA using recombinant YU2 ΔV1V2V3V5 gp120 protein (WT) or its mutated counterparts (W69A or R419D or the double mutant W69A/R419D). Serum binding to gp120 was detected using biotin-tagged dimeric rsFcγRIIIa (0.1 µg/ml) followed by the addition of HRP-conjugated streptavidin. Sera from 5 healthy individuals (HIV − ) were used as a negative control. Graphs represent relative light units (R.L.U.) determined from 3 independent experiments done in quadruplicate, with the error bars indicating means ± SEM. Statistical significance was tested using a paired t test or a Wilcoxon matched-pair signed-rank test (based on statistical normality) to compare HIV + serum data sets and a Mann-Whitney U test to compare HIV + sera with HIV − sera (****, P

    Journal: Journal of Virology

    Article Title: Two Families of Env Antibodies Efficiently Engage Fc-Gamma Receptors and Eliminate HIV-1-Infected Cells

    doi: 10.1128/JVI.01823-18

    Figure Lengend Snippet: Dimeric FcγRIIIa engagement by HIV + sera is driven by anti-CoRBS and anti-cluster A Abs. (A) Indirect ELISA using recombinant YU2 ΔV1V2V3V5 gp120 protein (0.25 µg/ml). gp120-coated wells were incubated with sera from 15 chronically HIV-1-infected individuals with or without A32 Fab and/or 17b Fab fragments (1 µg/ml). (B) Indirect ELISA using recombinant YU2 ΔV1V2V3V5 gp120 protein (WT) or its mutated counterparts (W69A or R419D or the double mutant W69A/R419D). Serum binding to gp120 was detected using biotin-tagged dimeric rsFcγRIIIa (0.1 µg/ml) followed by the addition of HRP-conjugated streptavidin. Sera from 5 healthy individuals (HIV − ) were used as a negative control. Graphs represent relative light units (R.L.U.) determined from 3 independent experiments done in quadruplicate, with the error bars indicating means ± SEM. Statistical significance was tested using a paired t test or a Wilcoxon matched-pair signed-rank test (based on statistical normality) to compare HIV + serum data sets and a Mann-Whitney U test to compare HIV + sera with HIV − sera (****, P

    Article Snippet: Horseradish peroxidase (HRP)-conjugated antibody specific for the Fc region of human IgG (Pierce) or high-sensitivity streptavidin-HRP (Thermo Fisher) (1/1,000 in blocking buffer) was added for 1 h at room temperature, followed by four washes.

    Techniques: Indirect ELISA, Recombinant, Incubation, Infection, Mutagenesis, Binding Assay, Negative Control, MANN-WHITNEY

    Anti-CoRBS and anti-cluster A Abs synergize to engage dimeric FcγRIIIa. Indirect ELISA was performed using recombinant full-length YU2 gp120 protein (0.4 µg/ml) (A and C) or using recombinant YU2 ΔV1V2V3V5 gp120 protein (0.25 µg/ml) (B and D). gp120-coated wells were incubated with a total concentration of 1 µg/ml of primary antibodies (A and C) with or without BNM-III-170 (25 µM). Antibody binding to gp120 was detected using biotin-tagged dimeric rsFcγRIIIa (0.1 µg/ml) followed by the addition of HRP-conjugated streptavidin (A and B) or using HRP-conjugated anti-human secondary antibody (C and D). Data in graphs represent relative light units (R.L.U.) determined from at least 5 independent experiments done in quadruplicate, with the error bars indicating means ± standard errors of the means (SEM). Statistical significance was tested using an unpaired t test or a Mann-Whitney U test based on statistical normality (*, P

    Journal: Journal of Virology

    Article Title: Two Families of Env Antibodies Efficiently Engage Fc-Gamma Receptors and Eliminate HIV-1-Infected Cells

    doi: 10.1128/JVI.01823-18

    Figure Lengend Snippet: Anti-CoRBS and anti-cluster A Abs synergize to engage dimeric FcγRIIIa. Indirect ELISA was performed using recombinant full-length YU2 gp120 protein (0.4 µg/ml) (A and C) or using recombinant YU2 ΔV1V2V3V5 gp120 protein (0.25 µg/ml) (B and D). gp120-coated wells were incubated with a total concentration of 1 µg/ml of primary antibodies (A and C) with or without BNM-III-170 (25 µM). Antibody binding to gp120 was detected using biotin-tagged dimeric rsFcγRIIIa (0.1 µg/ml) followed by the addition of HRP-conjugated streptavidin (A and B) or using HRP-conjugated anti-human secondary antibody (C and D). Data in graphs represent relative light units (R.L.U.) determined from at least 5 independent experiments done in quadruplicate, with the error bars indicating means ± standard errors of the means (SEM). Statistical significance was tested using an unpaired t test or a Mann-Whitney U test based on statistical normality (*, P

    Article Snippet: Horseradish peroxidase (HRP)-conjugated antibody specific for the Fc region of human IgG (Pierce) or high-sensitivity streptavidin-HRP (Thermo Fisher) (1/1,000 in blocking buffer) was added for 1 h at room temperature, followed by four washes.

    Techniques: Indirect ELISA, Recombinant, Incubation, Concentration Assay, Binding Assay, MANN-WHITNEY

    Introduction of LALA mutations in the Fc portion of both CoRBS and anti-cluster A antibodies decreases engagement of dimeric FcγRIIIa and ADCC against HIV-1-infected cells. (A and B) Indirect ELISA using recombinant YU2 ΔV1V2V3V5 gp120 protein (0.25 µg/ml) with a total concentration of 1 µg/ml of primary antibodies. Antibody binding to gp120 was detected using (A) a biotin-tagged dimeric rsFcγRIIIa (0.1 µg/ml) followed by the addition of HRP-conjugated streptavidin or using (B) HRP-conjugated anti-human secondary antibody. (A and B) Graphs represent relative light units (R.L.U.) determined from at least 5 independent experiments done in quadruplicate, with the error bars indicating means ± SEM. (C and D) Cell surface staining of primary CD4 + T cells infected with CH58 T/F virus strains that were either (C) WT or (D) defective for Nef and Vpu expression (N-U-) with a total concentration of 2.5 µg/ml of primary antibodies. (C) Staining with CH58 T/F WT virus was done in the presence of BNM-III-170 (50 µM). (C and D) Antibody binding was detected using a biotin-tagged dimeric rsFcγRIIIa (0.2 µg/ml) followed by the addition of Alexa Fluor 647-conjugated streptavidin. Graphs represent mean fluorescence intensities (MFI) in the infected (p24 + ) population determined from 5 independent experiments, with the error bars indicating means ± SEM. (E and F) Primary CD4 + ). (E) Analysis of ADCC with CH58 T/F WT was done in the presence of BNM-III-170 (50 µM). Shown in panels E and F are the percentages of ADCC-mediated killing obtained with a total concentration of 2.5 µg/ml of antibodies. Data are representative of results from 5 independent experiments, with the error bars indicating means ± SEM. Statistical significance was tested using an unpaired t test or a Mann-Whitney U test based on statistical normality (*, P

    Journal: Journal of Virology

    Article Title: Two Families of Env Antibodies Efficiently Engage Fc-Gamma Receptors and Eliminate HIV-1-Infected Cells

    doi: 10.1128/JVI.01823-18

    Figure Lengend Snippet: Introduction of LALA mutations in the Fc portion of both CoRBS and anti-cluster A antibodies decreases engagement of dimeric FcγRIIIa and ADCC against HIV-1-infected cells. (A and B) Indirect ELISA using recombinant YU2 ΔV1V2V3V5 gp120 protein (0.25 µg/ml) with a total concentration of 1 µg/ml of primary antibodies. Antibody binding to gp120 was detected using (A) a biotin-tagged dimeric rsFcγRIIIa (0.1 µg/ml) followed by the addition of HRP-conjugated streptavidin or using (B) HRP-conjugated anti-human secondary antibody. (A and B) Graphs represent relative light units (R.L.U.) determined from at least 5 independent experiments done in quadruplicate, with the error bars indicating means ± SEM. (C and D) Cell surface staining of primary CD4 + T cells infected with CH58 T/F virus strains that were either (C) WT or (D) defective for Nef and Vpu expression (N-U-) with a total concentration of 2.5 µg/ml of primary antibodies. (C) Staining with CH58 T/F WT virus was done in the presence of BNM-III-170 (50 µM). (C and D) Antibody binding was detected using a biotin-tagged dimeric rsFcγRIIIa (0.2 µg/ml) followed by the addition of Alexa Fluor 647-conjugated streptavidin. Graphs represent mean fluorescence intensities (MFI) in the infected (p24 + ) population determined from 5 independent experiments, with the error bars indicating means ± SEM. (E and F) Primary CD4 + ). (E) Analysis of ADCC with CH58 T/F WT was done in the presence of BNM-III-170 (50 µM). Shown in panels E and F are the percentages of ADCC-mediated killing obtained with a total concentration of 2.5 µg/ml of antibodies. Data are representative of results from 5 independent experiments, with the error bars indicating means ± SEM. Statistical significance was tested using an unpaired t test or a Mann-Whitney U test based on statistical normality (*, P

    Article Snippet: Horseradish peroxidase (HRP)-conjugated antibody specific for the Fc region of human IgG (Pierce) or high-sensitivity streptavidin-HRP (Thermo Fisher) (1/1,000 in blocking buffer) was added for 1 h at room temperature, followed by four washes.

    Techniques: Infection, Indirect ELISA, Recombinant, Concentration Assay, Binding Assay, Staining, Expressing, Fluorescence, MANN-WHITNEY

    Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. SAv-HRP, streptavidin–horseradish peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3

    Journal: Nature Communications

    Article Title: Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide

    doi: 10.1038/s41467-018-04343-0

    Figure Lengend Snippet: Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. SAv-HRP, streptavidin–horseradish peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3

    Article Snippet: The samples were analysed by western blotting using Streptavidin-HRP conjugate (SAv-HRP, Thermo, S911, 1:5000) and Coomassie Brilliant Blue (CBB) stain.

    Techniques: Western Blot, Recombinant, Incubation, Staining, Concentration Assay, Fluorescence

    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Journal: ACS nano

    Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter

    doi: 10.1021/nn404945r

    Figure Lengend Snippet: GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Article Snippet: Gluc or GlucB labeled EVs in PBS were spotted onto nitrocellulose membranes at 16, 31, 63, 125, 250, 500 and 1,000 ng protein followed by overnight incubation in 5% BSA fraction V (Invitrogen), immunoblotting with streptavidin-HRP conjugate (Pierce) and chemiluminescence detection of biotinylated EVs with SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) on autoradiography films (Denville Scientific, Metuchen, NJ).

    Techniques: Expressing, Live Cell Imaging, Stable Transfection, Transduction, Western Blot, Negative Control, Transmission Assay, Transmission Electron Microscopy, Labeling, Immunolabeling, Dot Blot, Isolation

    Identification of in vivo binding partners of AKAP95. (A) Schematic representation of Myc-BirA*-AKAP95. (B) Western blot analysis using anti-myc antibody and HRP-labeled streptavidin of lysates from Myc-BirA*-AKAP95 expressing- and control U2OS cells cultured as indicated. (C) Immunolocalization of Myc-BirA*-AKAP95 (anti-myc; red) and protein biotinylation (FITC-labeled streptavidin; green) in stable Myc-BirA*–AKAP95 U2OS cells cultured as indicated. Immunolocalization of AKAP95 in U2OS cells (bottom). Scale bar, 5 μm. (D) Gene ontology analysis and nuclear component classification of proteins identified by AKAP95 BioID. (E) Western blot analysis using indicated antibodies of the samples from Myc-BirA*-AKAP95-expressing and control U2OS cells that were subjected to BioID.

    Journal: Cell Cycle

    Article Title: AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint

    doi: 10.1080/15384101.2017.1310350

    Figure Lengend Snippet: Identification of in vivo binding partners of AKAP95. (A) Schematic representation of Myc-BirA*-AKAP95. (B) Western blot analysis using anti-myc antibody and HRP-labeled streptavidin of lysates from Myc-BirA*-AKAP95 expressing- and control U2OS cells cultured as indicated. (C) Immunolocalization of Myc-BirA*-AKAP95 (anti-myc; red) and protein biotinylation (FITC-labeled streptavidin; green) in stable Myc-BirA*–AKAP95 U2OS cells cultured as indicated. Immunolocalization of AKAP95 in U2OS cells (bottom). Scale bar, 5 μm. (D) Gene ontology analysis and nuclear component classification of proteins identified by AKAP95 BioID. (E) Western blot analysis using indicated antibodies of the samples from Myc-BirA*-AKAP95-expressing and control U2OS cells that were subjected to BioID.

    Article Snippet: Biotinylated proteins were detected directly using HRP-Streptavidin conjugate (Invitrogen; 43-4323; 1:5000).

    Techniques: In Vivo, Binding Assay, Western Blot, Labeling, Expressing, Cell Culture

    Amplified microscopic detection of PTHR 1 s using the ligand PTH-HRP. The primary reaction of this ligand was with biotin-phenol (“protocol B”); the secondary detection with either streptavidin-HRP + TrueBlue TM or streptavidin-Qdots. These schemes were applied to signals generated by PTH-HRP either in transfected HEK 293a cells or in HOS cells. Transmission and epifluorescence, original magnification: 100 × (TrueBlue™) or 1000 × (Qdots).

    Journal: Scientific Reports

    Article Title: Production and evaluation of parathyroid hormone receptor1 ligands with intrinsic or assembled peroxidase domains

    doi: 10.1038/s41598-017-13548-0

    Figure Lengend Snippet: Amplified microscopic detection of PTHR 1 s using the ligand PTH-HRP. The primary reaction of this ligand was with biotin-phenol (“protocol B”); the secondary detection with either streptavidin-HRP + TrueBlue TM or streptavidin-Qdots. These schemes were applied to signals generated by PTH-HRP either in transfected HEK 293a cells or in HOS cells. Transmission and epifluorescence, original magnification: 100 × (TrueBlue™) or 1000 × (Qdots).

    Article Snippet: Then, the cells were incubated 30 minutes with a HBSS solution containing 1% BSA to block the non-specific binding sites before being incubated with a horse radish peroxidase conjugate of streptavidin (SA-HRP; Invitrogen) for 1 hour.

    Techniques: Amplification, Generated, Transfection, Transmission Assay

    TrueBlue TM labeling of PTHR 1 expressing cells following stimulation with PTH-APEX2. Protocol A: HEK 293a cells transfected with either pcDNA3.1 or PTHR 1 were stimulated for 30 minutes with PTH-APEX2 followed by a staining with TrueBlue TM . Optionally, cells were co-stimulated with PTH 1–34 to evaluate the specificity of PTH-APEX2. The cells were observed in light transmission (final magnification 100×). Protocol B: a biotinyl-phenol signal amplification step followed incubation of cells with the receptor ligands, and a final incubation with streptavidin-HRP supported the staining with TrueBlue™, as outlined in Methods.

    Journal: Scientific Reports

    Article Title: Production and evaluation of parathyroid hormone receptor1 ligands with intrinsic or assembled peroxidase domains

    doi: 10.1038/s41598-017-13548-0

    Figure Lengend Snippet: TrueBlue TM labeling of PTHR 1 expressing cells following stimulation with PTH-APEX2. Protocol A: HEK 293a cells transfected with either pcDNA3.1 or PTHR 1 were stimulated for 30 minutes with PTH-APEX2 followed by a staining with TrueBlue TM . Optionally, cells were co-stimulated with PTH 1–34 to evaluate the specificity of PTH-APEX2. The cells were observed in light transmission (final magnification 100×). Protocol B: a biotinyl-phenol signal amplification step followed incubation of cells with the receptor ligands, and a final incubation with streptavidin-HRP supported the staining with TrueBlue™, as outlined in Methods.

    Article Snippet: Then, the cells were incubated 30 minutes with a HBSS solution containing 1% BSA to block the non-specific binding sites before being incubated with a horse radish peroxidase conjugate of streptavidin (SA-HRP; Invitrogen) for 1 hour.

    Techniques: Labeling, Expressing, Transfection, Staining, Transmission Assay, Amplification, Incubation

    Schematic representation of the design of fusion proteins evaluated as potential PTHR 1 ligands. The fusion proteins evaluated in this paper consist of the full sequence of prepro-PTH 1–84 fused to the N-terminus of either a peroxidase (PTH-APEX2 or PTH-HRP) or an antigenic domain containing two epitopes (PTH-myc) recognized by commercially available monoclonal antibodies. The peroxidase domains, either covalently bound to PTH or assembled through antibodies, were detected using either a colorimetric substrate (TrueBlue™ or TMB), a luminescent substrate (luminol) or, alternatively, biotin-phenol or biotinyl-tyramide, peroxidase substrates whose oxidized states form covalent bonds leading to a proximal accumulation of biotin which can be detected by conjugated streptavidin molecules.

    Journal: Scientific Reports

    Article Title: Production and evaluation of parathyroid hormone receptor1 ligands with intrinsic or assembled peroxidase domains

    doi: 10.1038/s41598-017-13548-0

    Figure Lengend Snippet: Schematic representation of the design of fusion proteins evaluated as potential PTHR 1 ligands. The fusion proteins evaluated in this paper consist of the full sequence of prepro-PTH 1–84 fused to the N-terminus of either a peroxidase (PTH-APEX2 or PTH-HRP) or an antigenic domain containing two epitopes (PTH-myc) recognized by commercially available monoclonal antibodies. The peroxidase domains, either covalently bound to PTH or assembled through antibodies, were detected using either a colorimetric substrate (TrueBlue™ or TMB), a luminescent substrate (luminol) or, alternatively, biotin-phenol or biotinyl-tyramide, peroxidase substrates whose oxidized states form covalent bonds leading to a proximal accumulation of biotin which can be detected by conjugated streptavidin molecules.

    Article Snippet: Then, the cells were incubated 30 minutes with a HBSS solution containing 1% BSA to block the non-specific binding sites before being incubated with a horse radish peroxidase conjugate of streptavidin (SA-HRP; Invitrogen) for 1 hour.

    Techniques: Sequencing

    Schematic of an amplification strategy using streptavidin poly-HRP. The streptavidin poly-HRP is attached to the biotinylated anti-human IL-6 detection antibody.

    Journal: Lab on a Chip

    Article Title: Fabrication of immunosensor microwell arrays from gold compact discs for detection of cancer biomarker proteins †

    doi: 10.1039/c1lc20833k

    Figure Lengend Snippet: Schematic of an amplification strategy using streptavidin poly-HRP. The streptavidin poly-HRP is attached to the biotinylated anti-human IL-6 detection antibody.

    Article Snippet: Streptavidin poly-HRP was from Thermo Scientific (product # N200).

    Techniques: Amplification

    Oligonucleotides identified by ADAPT reveal aptamer-like characteristics. ( a ) Filter retention analysis of C1Q-binding by the ssODNs H1 (dark grey columns) and H11 (pale grey columns) at indicated C1Q concentrations. As a control, the reverse complement of H1, H1RC, was used (black columns). ( b ) ELONA analysis of C1Q-binding by the ssODNs H11 (circles) at indicated concentrations and a fixed C1Q concentration (0.625 nM). As a control, the reverse complement of H11, H11RC, was used (squares). “No aptamer” control (triangles) shows low background binding of detector Streptavidin-HRP. H11 specifically binds C1Q (estimated K D around 40 nM). ( c ) PAGE analysis of PEG-precipitated and ssODN-associated proteins pulled-down with L2. Lane 1: Molecular weight marker; lane 2: Input library L2; lane 3: Fraction pulled down by biotinylated L2; lane 4: Fraction pulled down by non-biotinylated L2; lane 5: Fraction found in the absence of DNA library; red arrows indicate the ssODN library; yellow arrows indicate protein bands cut out and analysed by LC-MS/MS; black arrows indicate streptavidin monomers leaking from beads. ( d ) Four-way Venn diagram of proteins detected by LC-MS/MS from Unfractionated plasma, PEG-precipitated plasma, PEG-precipitated plasma in presence of L0 or L2, respectively, purified by streptavidin magnetic beads (background-subtracted; i.e. biotinylated minus non-biotinylated libraries). ( e ) Gene ontology (GO) cellular component enrichment analysis of the subset of proteins associated with L0 (red bars) and L2 (grey bars) that show a p value of at least 6 × 10 −12 . Proteins listed are: 1 extracellular region part, 2 extracellular region, 3 extracellular exosome, 4 extracellular membrane-bounded organelle, 5 extracellular organelle, 6 extracellular vesicle, 7 membrane-bounded vesicle, 8 vesicle, 9 organelle, 10 membrane-bounded organelle, 11 extracellular space, 12 focal adhesion, 13 cell-substrate adherence junction, 14 cell-substrate junction, 15 adherence junction, 16 anchoring junction, 17 cellular component, 18 cell junction, 19 blood micro particle. Inset: 108 proteins pulled down by L2 (inset, 96 + 12), cut from the gel shown in ( c ), and analysed by LC-MS/MS, 13 proteins unique to background-subtracted L0 (inset, 13), and 12 overlapping proteins (inset, 12).

    Journal: Scientific Reports

    Article Title: Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach

    doi: 10.1038/srep42741

    Figure Lengend Snippet: Oligonucleotides identified by ADAPT reveal aptamer-like characteristics. ( a ) Filter retention analysis of C1Q-binding by the ssODNs H1 (dark grey columns) and H11 (pale grey columns) at indicated C1Q concentrations. As a control, the reverse complement of H1, H1RC, was used (black columns). ( b ) ELONA analysis of C1Q-binding by the ssODNs H11 (circles) at indicated concentrations and a fixed C1Q concentration (0.625 nM). As a control, the reverse complement of H11, H11RC, was used (squares). “No aptamer” control (triangles) shows low background binding of detector Streptavidin-HRP. H11 specifically binds C1Q (estimated K D around 40 nM). ( c ) PAGE analysis of PEG-precipitated and ssODN-associated proteins pulled-down with L2. Lane 1: Molecular weight marker; lane 2: Input library L2; lane 3: Fraction pulled down by biotinylated L2; lane 4: Fraction pulled down by non-biotinylated L2; lane 5: Fraction found in the absence of DNA library; red arrows indicate the ssODN library; yellow arrows indicate protein bands cut out and analysed by LC-MS/MS; black arrows indicate streptavidin monomers leaking from beads. ( d ) Four-way Venn diagram of proteins detected by LC-MS/MS from Unfractionated plasma, PEG-precipitated plasma, PEG-precipitated plasma in presence of L0 or L2, respectively, purified by streptavidin magnetic beads (background-subtracted; i.e. biotinylated minus non-biotinylated libraries). ( e ) Gene ontology (GO) cellular component enrichment analysis of the subset of proteins associated with L0 (red bars) and L2 (grey bars) that show a p value of at least 6 × 10 −12 . Proteins listed are: 1 extracellular region part, 2 extracellular region, 3 extracellular exosome, 4 extracellular membrane-bounded organelle, 5 extracellular organelle, 6 extracellular vesicle, 7 membrane-bounded vesicle, 8 vesicle, 9 organelle, 10 membrane-bounded organelle, 11 extracellular space, 12 focal adhesion, 13 cell-substrate adherence junction, 14 cell-substrate junction, 15 adherence junction, 16 anchoring junction, 17 cellular component, 18 cell junction, 19 blood micro particle. Inset: 108 proteins pulled down by L2 (inset, 96 + 12), cut from the gel shown in ( c ), and analysed by LC-MS/MS, 13 proteins unique to background-subtracted L0 (inset, 13), and 12 overlapping proteins (inset, 12).

    Article Snippet: Biotinylated ssODN was pre-incubated with streptavidin-poly HRP (Pierce, Waltham, USA) for 15 min at 22 °C.

    Techniques: Binding Assay, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight, Marker, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Purification, Magnetic Beads

    Chronic ethanol feeding increased oxidative stress in adipocytes and adipose tissue. (A) 4-HNE protein adducts were quantified by slot blot analysis in isolated epididymal adipocytes. n=7. (B) Proteins were extracted from rat epididymal adipose tissue, coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with HRP-streptavidin. HSC70 was used as loading control. (C) Results of densitometry after normalization to HSC70. Values represent means ± SEM. n=4. Values with an asterisk are significantly different from each other, p

    Journal: Alcoholism, Clinical and Experimental Research

    Article Title: Ethanol-induced oxidative stress via the CYP2E1 pathway disrupts adiponectin secretion from adipocytes

    doi: 10.1111/j.1530-0277.2011.01607.x

    Figure Lengend Snippet: Chronic ethanol feeding increased oxidative stress in adipocytes and adipose tissue. (A) 4-HNE protein adducts were quantified by slot blot analysis in isolated epididymal adipocytes. n=7. (B) Proteins were extracted from rat epididymal adipose tissue, coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with HRP-streptavidin. HSC70 was used as loading control. (C) Results of densitometry after normalization to HSC70. Values represent means ± SEM. n=4. Values with an asterisk are significantly different from each other, p

    Article Snippet: EZ-link biotin hydrazide and poly-HRP streptavidin were purchased from Pierce (Rockford, IL).

    Techniques: Dot Blot, Isolation, SDS Page

    Characterization of ZASP-skeletal muscle α-actin 1 (ACTA1) interaction in mammalian cells and in vitro with purified proteins. A , co-IP ( IP ) assay showing the interaction between the ZASP isoforms and ACTA1 in wild-type mouse vastus muscle lysates. B and C , co-IP assays showing the interaction between the indicated ZASP isoforms and ACTA1 in non-muscle (COS7/HEK293) cells. The proteins were detected with anti-FLAG and anti-HA antibodies. D , purified GST-tagged ZASP-LΔex10 (WT, A165V, and A147T), but not GST alone, binds to biotinylated G-actin ( G-actin ) in a slot blot overlay assay. Biotin-G-actin was detected with HRP-streptavidin. GST and GST-ZASP were detected by immunoblotting with GST antibody ( anti-GST ). Arp2/3 and BSA served as positive and negative controls for G-actin binding. E , high-speed cosedimentation assay of purified GST or the indicated GST-ZASP-LΔex10 proteins and F-actin. Representative colloidal blue-stained gel images demonstrate a shift of the indicated GST-ZASP proteins from the supernatant ( S ) to the pellet ( P ) fraction in the presence of F-actin, whereas GST remained in the supernatant. Nonlinear regression analysis of the ZASP-actin interaction is shown in the right panel (triplicate assays, data represent mean ± S.D.).

    Journal: The Journal of Biological Chemistry

    Article Title: Z-disc-associated, Alternatively Spliced, PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy *

    doi: 10.1074/jbc.M114.550418

    Figure Lengend Snippet: Characterization of ZASP-skeletal muscle α-actin 1 (ACTA1) interaction in mammalian cells and in vitro with purified proteins. A , co-IP ( IP ) assay showing the interaction between the ZASP isoforms and ACTA1 in wild-type mouse vastus muscle lysates. B and C , co-IP assays showing the interaction between the indicated ZASP isoforms and ACTA1 in non-muscle (COS7/HEK293) cells. The proteins were detected with anti-FLAG and anti-HA antibodies. D , purified GST-tagged ZASP-LΔex10 (WT, A165V, and A147T), but not GST alone, binds to biotinylated G-actin ( G-actin ) in a slot blot overlay assay. Biotin-G-actin was detected with HRP-streptavidin. GST and GST-ZASP were detected by immunoblotting with GST antibody ( anti-GST ). Arp2/3 and BSA served as positive and negative controls for G-actin binding. E , high-speed cosedimentation assay of purified GST or the indicated GST-ZASP-LΔex10 proteins and F-actin. Representative colloidal blue-stained gel images demonstrate a shift of the indicated GST-ZASP proteins from the supernatant ( S ) to the pellet ( P ) fraction in the presence of F-actin, whereas GST remained in the supernatant. Nonlinear regression analysis of the ZASP-actin interaction is shown in the right panel (triplicate assays, data represent mean ± S.D.).

    Article Snippet: The membrane was incubated with HRP-conjugated streptavidin (N100, Thermo Scientific), and G-actin binding was detected using chemiluminescence.

    Techniques: In Vitro, Purification, Co-Immunoprecipitation Assay, Dot Blot, Overlay Assay, Binding Assay, Staining

    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.

    Journal: Retrovirology

    Article Title: Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses

    doi: 10.1186/1742-4690-5-2

    Figure Lengend Snippet: Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.

    Article Snippet: The membrane was stripped using Pierce Stripping Buffer (catalog no. 21059) and reprobed using HRP-conjugated streptavidin (Pierce; catalog no. 21126).

    Techniques: Expressing

    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

    Early disease onset correlates with increased stability of mutant TDP-43 proteins in familial ALS. A , schematic drawing of human TDP-43 and its mutations. ALS-linked mutations of TDP-43 from familial ( upper ) and sporadic ( lower ) ALS cases as well as the locations of the NLS and NES are shown. The mutations used in our study are shown in boldface type (almost all familial ALS-linked mutations), and mutations with more than four patients are shown in red . The epitopes recognized by the anti-TDP-43 antibodies used in this study are indicated. B and C , half-lives of wild-type and mutant TDP-43 proteins. B , transfected Neuro2a cells were metabolically labeled with the methionine analog AHA. Labeled TDP-43 was immunoprecipitated and visualized by HRP-streptavidin as described under “Experimental Procedures.” C , labeled TDP-43 bands were quantified, and the averages of three independent experiments were plotted. Half-lives of wild-type and mutant TDP-43 proteins were calculated by curve-fitting using this graph ( B, right ). Error bars represent S.E. D and E , half-lives of familial ALS-linked mutant TDP-43 proteins were negatively correlated with age of disease onset but not disease duration. Calculated half-lives of TDP-43 proteins were plotted against mean ages of disease onset ( D ) or duration ( E ). The correlations between each parameter and age of onset or duration were evaluated by the correlation coefficient ( r ) and probability ( p ) shown in each graph ( D and E ).

    Journal: The Journal of Biological Chemistry

    Article Title: Accelerated Disease Onset with Stabilized Familial Amyotrophic Lateral Sclerosis (ALS)-linked Mutant TDP-43 Proteins *

    doi: 10.1074/jbc.M112.433615

    Figure Lengend Snippet: Early disease onset correlates with increased stability of mutant TDP-43 proteins in familial ALS. A , schematic drawing of human TDP-43 and its mutations. ALS-linked mutations of TDP-43 from familial ( upper ) and sporadic ( lower ) ALS cases as well as the locations of the NLS and NES are shown. The mutations used in our study are shown in boldface type (almost all familial ALS-linked mutations), and mutations with more than four patients are shown in red . The epitopes recognized by the anti-TDP-43 antibodies used in this study are indicated. B and C , half-lives of wild-type and mutant TDP-43 proteins. B , transfected Neuro2a cells were metabolically labeled with the methionine analog AHA. Labeled TDP-43 was immunoprecipitated and visualized by HRP-streptavidin as described under “Experimental Procedures.” C , labeled TDP-43 bands were quantified, and the averages of three independent experiments were plotted. Half-lives of wild-type and mutant TDP-43 proteins were calculated by curve-fitting using this graph ( B, right ). Error bars represent S.E. D and E , half-lives of familial ALS-linked mutant TDP-43 proteins were negatively correlated with age of disease onset but not disease duration. Calculated half-lives of TDP-43 proteins were plotted against mean ages of disease onset ( D ) or duration ( E ). The correlations between each parameter and age of onset or duration were evaluated by the correlation coefficient ( r ) and probability ( p ) shown in each graph ( D and E ).

    Article Snippet: Following immunoprecipitation using an anti-TDP-43 C-terminal antibody under denaturing conditions, AHA-labeled TDP-43 protein was visualized by high affinity HRP-conjugated streptavidin (Thermo Scientific).

    Techniques: Mutagenesis, Transfection, Metabolic Labelling, Labeling, Immunoprecipitation

    Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific streptavidin binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase (HRP), streptavidin (SA), primary antibody (1° Ab)

    Journal: Journal of immunological methods

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES

    doi: 10.1016/j.jim.2018.10.006

    Figure Lengend Snippet: Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific streptavidin binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase (HRP), streptavidin (SA), primary antibody (1° Ab)

    Article Snippet: In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential.

    Techniques: Binding Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Cytometry

    BioITC inhibits MEKK1, but not the C1238V mutant, by covalent modification . A . Purified MEKK1 or the C1238V mutant on chitin beads were treated with Bio-ITC at the concentrations indicated in 50 mM Tris pH7.8 for 40 minutes at room temperature. Bio-ITC was removed, and the kinase activity measured. Bio-ITC inhibits wild type MEKK1, but not the C1238V mutant in a dose dependent manner. B . Purified wild type MEKK1 or the C1238V mutant were treated as in Panel A with Bio-ITC at the indicated concentrations. The treated protein was used to either assay for kinase activity (top panel) or to detect covalent modification by Bio-ITC using a streptavidin-HRP conjugate (middle panel). Equivalent protein was confirmed by re-probing the streptavidin gel with anti-MEKK1 (bottom panel), using an alkaline phosphatase conjugated secondary antibody and colorimetric detection reagents. Bio-ITC inhibited wild type MEKK1 by stable, covalent modification in a manner that requires C1238V. C . CV-1 cells expressing either wild type MEKK1 or the C1238V mutant were lysed in Tris lysis buffer containing 0.1% NP-40. Clarified lysates were treated by addition of either PEITC (P) or Bio-ITC (B) to final concentration of 250 μM, and incubated at room temperature for 30 minutes. MEKK1 proteins were purified on chitin beads and kinase activity measured. Both PEITC and Bio-ITC completely inhibited wild type MEKK1, but not the C1238V mutant when added to cell lysates.

    Journal: BMC Cancer

    Article Title: The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

    doi: 10.1186/1471-2407-7-183

    Figure Lengend Snippet: BioITC inhibits MEKK1, but not the C1238V mutant, by covalent modification . A . Purified MEKK1 or the C1238V mutant on chitin beads were treated with Bio-ITC at the concentrations indicated in 50 mM Tris pH7.8 for 40 minutes at room temperature. Bio-ITC was removed, and the kinase activity measured. Bio-ITC inhibits wild type MEKK1, but not the C1238V mutant in a dose dependent manner. B . Purified wild type MEKK1 or the C1238V mutant were treated as in Panel A with Bio-ITC at the indicated concentrations. The treated protein was used to either assay for kinase activity (top panel) or to detect covalent modification by Bio-ITC using a streptavidin-HRP conjugate (middle panel). Equivalent protein was confirmed by re-probing the streptavidin gel with anti-MEKK1 (bottom panel), using an alkaline phosphatase conjugated secondary antibody and colorimetric detection reagents. Bio-ITC inhibited wild type MEKK1 by stable, covalent modification in a manner that requires C1238V. C . CV-1 cells expressing either wild type MEKK1 or the C1238V mutant were lysed in Tris lysis buffer containing 0.1% NP-40. Clarified lysates were treated by addition of either PEITC (P) or Bio-ITC (B) to final concentration of 250 μM, and incubated at room temperature for 30 minutes. MEKK1 proteins were purified on chitin beads and kinase activity measured. Both PEITC and Bio-ITC completely inhibited wild type MEKK1, but not the C1238V mutant when added to cell lysates.

    Article Snippet: The Streptavidin-HRP detection reagent was from Zymed.

    Techniques: Mutagenesis, Modification, Purification, Activity Assay, Expressing, Lysis, Concentration Assay, Incubation

    Covalent modification of MEKK1 by Bio-ITC is blocked by preincubation of cells with PEITC . CV-1 cells expressing wild type MEKK1 or the C1238V mutant were either left untreated or incubated with 250 μM PEITC for 20 minutes at 37°C. Lysates were prepared in TLB, clarified and then 500 μM Bio-ITC was added to the samples as indicated. MEKK1 proteins were purified from the lysates on chitin beads, and either electrophoresed for detection of biotin labeling using streptavidin-HRP (top panel) or assayed for kinase activity (middle panel). To confirm equivalent protein, the SA-HRP blot was re-probed with anti-MEKK1 as in Figure 5. Lane 2 contains the molecular weight markers. Bio-ITC covalently modifies MEKK1 protein and inhibits its activity in a manner that requires C1238V. The modification by Bio-ITC is completely inhibited by preincubation of the cells with PEITC, suggesting that the modification site is completely occupied by the natural isothiocyanate after exposure of intact cells.

    Journal: BMC Cancer

    Article Title: The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

    doi: 10.1186/1471-2407-7-183

    Figure Lengend Snippet: Covalent modification of MEKK1 by Bio-ITC is blocked by preincubation of cells with PEITC . CV-1 cells expressing wild type MEKK1 or the C1238V mutant were either left untreated or incubated with 250 μM PEITC for 20 minutes at 37°C. Lysates were prepared in TLB, clarified and then 500 μM Bio-ITC was added to the samples as indicated. MEKK1 proteins were purified from the lysates on chitin beads, and either electrophoresed for detection of biotin labeling using streptavidin-HRP (top panel) or assayed for kinase activity (middle panel). To confirm equivalent protein, the SA-HRP blot was re-probed with anti-MEKK1 as in Figure 5. Lane 2 contains the molecular weight markers. Bio-ITC covalently modifies MEKK1 protein and inhibits its activity in a manner that requires C1238V. The modification by Bio-ITC is completely inhibited by preincubation of the cells with PEITC, suggesting that the modification site is completely occupied by the natural isothiocyanate after exposure of intact cells.

    Article Snippet: The Streptavidin-HRP detection reagent was from Zymed.

    Techniques: Modification, Expressing, Mutagenesis, Incubation, Purification, Labeling, Activity Assay, Molecular Weight

    Aging-associated change on ER expression and DNA methylation. ( A ) mRNA expression for ERα and ERβ in mice aorta normalized to the 18S expression. ( B ) Image on top shows a representative experiment of biotin-dCTP incorporation to DNA following no digestion (Mock) and digestion with MspI (no methylation sensitive) or HpaII (methylation sensitive).DNA was spotted in a nylon membrane and visualized with streptavidin-HRP method (data shown in triplicate). Bar graphs show (left) the results of densitometric analyses from pooled data for global DNA methylation and (right) percentage of specific DNA methylation at 5′ flanking region of the gene encoding ERα. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p

    Journal: PLoS ONE

    Article Title: Aging Negatively Affects Estrogens-Mediated Effects on Nitric Oxide Bioavailability by Shifting ER?/ER? Balance in Female Mice

    doi: 10.1371/journal.pone.0025335

    Figure Lengend Snippet: Aging-associated change on ER expression and DNA methylation. ( A ) mRNA expression for ERα and ERβ in mice aorta normalized to the 18S expression. ( B ) Image on top shows a representative experiment of biotin-dCTP incorporation to DNA following no digestion (Mock) and digestion with MspI (no methylation sensitive) or HpaII (methylation sensitive).DNA was spotted in a nylon membrane and visualized with streptavidin-HRP method (data shown in triplicate). Bar graphs show (left) the results of densitometric analyses from pooled data for global DNA methylation and (right) percentage of specific DNA methylation at 5′ flanking region of the gene encoding ERα. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p

    Article Snippet: After washing with 0.4 N NaOH and cross-link at U.V. light, biotin incorporated to the membranes was visualized with HRP-streptavidin reaction (Pierce), according to manufacturer.

    Techniques: Expressing, DNA Methylation Assay, Mouse Assay, Methylation, Derivative Assay

    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).

    Journal: The Journal of General Physiology

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

    doi: 10.1085/jgp.200409031

    Figure Lengend 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).

    Article Snippet: Depending on the experiment, the nitrocellulose was probed with (a) primary antibody (1/1,000 dilution) followed by horseradish peroxidase–conjugated goat anti–rabbit IgG (1/7,000) (Jackson ImmunoResearch Laboratories) in PBS-T with 1% dry milk or (b) horseradish peroxidase–conjugated streptavidin (Pierce Chemical Co.).

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

    Parp-1 binds to the BRCA2 promoter. A , MCF-7 cell nuclear extracts were mixed with a biotinylated WT probe and a mutant probe ( M ). The DNA-protein complex was isolated with streptavidin-labeled magnetic beads. The magnetic bead column was washed and eluates were collected. Eluted fractions were separated on 10% acrylamide gels and visualized by silver staining. The 120-kDa protein band indicates a prominent DNA-protein complex. BSA was used as a control, and its position is indicated by a black arrow . The experiment was carried out three times. E1, E2, E3 (WT probe eluates); M , mutant probe eluate. B , mass spectrometry of the ∼120-kDa protein. C , EMSAs and antibody super-shifts were performed in MCF-7 nuclear extracts using WT, mutant probes, and Parp-1 antibody. D , EMSAs were carried out in MCF-7 nuclear extracts using the end-streptavidin blocked-biotin-labeled WT and mutant probes. E , luciferase activity of Del-9 in MCF-7 cells treated with 3-AB/NU1025, two Parp-1 inhibitors, or DMSO for 12 h.

    Journal: The Journal of Biological Chemistry

    Article Title: Poly(ADP-ribose) Polymerase-1 Down-regulates BRCA2 Expression through theBRCA2 Promoter *

    doi: 10.1074/jbc.M803693200

    Figure Lengend Snippet: Parp-1 binds to the BRCA2 promoter. A , MCF-7 cell nuclear extracts were mixed with a biotinylated WT probe and a mutant probe ( M ). The DNA-protein complex was isolated with streptavidin-labeled magnetic beads. The magnetic bead column was washed and eluates were collected. Eluted fractions were separated on 10% acrylamide gels and visualized by silver staining. The 120-kDa protein band indicates a prominent DNA-protein complex. BSA was used as a control, and its position is indicated by a black arrow . The experiment was carried out three times. E1, E2, E3 (WT probe eluates); M , mutant probe eluate. B , mass spectrometry of the ∼120-kDa protein. C , EMSAs and antibody super-shifts were performed in MCF-7 nuclear extracts using WT, mutant probes, and Parp-1 antibody. D , EMSAs were carried out in MCF-7 nuclear extracts using the end-streptavidin blocked-biotin-labeled WT and mutant probes. E , luciferase activity of Del-9 in MCF-7 cells treated with 3-AB/NU1025, two Parp-1 inhibitors, or DMSO for 12 h.

    Article Snippet: The biotinylated double-strand probes were modified by binding horseradish peroxidase-conjugated streptavidin (Pierce) to the DNA ends according to the manufacturer's instructions.

    Techniques: Mutagenesis, Isolation, Labeling, Magnetic Beads, Silver Staining, Mass Spectrometry, Luciferase, Activity Assay