streptavidin -- horseradish Search Results


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  • 99
    Vector Laboratories streptavidin horseradish peroxidease
    Streptavidin Horseradish Peroxidease, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin
    Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 8270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cusabio horseradish streptavidin
    Horseradish Streptavidin, supplied by Cusabio, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology horseradish streptavidin
    Horseradish Streptavidin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish conjugate
    Streptavidin Horseradish Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies horseradish conjugated streptavidin
    Horseradish Conjugated Streptavidin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson horseradish conjugated streptavidin
    Horseradish Conjugated Streptavidin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology horseradish labeled streptavidin
    Horseradish Labeled Streptavidin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega streptavidin horseradish peroxidase
    Dia-1 siRNA blocks RAGE ligand-stimulated cellular migration. C6 glioma cells stably expressing RAGE were transfected with siRNAs against Dia-1 or siRNA control. 48 h after transfection cells were lysed, and Dia-1 levels were assessed by quantitative-PCR ( A ) or Western blotting ( WB ) using anti-Dia-1 antibodies ( B ). C , cells were transfected with either Dia-1 siRNA/GFP and scramble siRNA/GFP and, after 48 h, fixed and immunostained with antibodies to Dia-1 followed by respective biotinylated secondary antibodies and <t>streptavidin-linked</t> Alexa-546 (Dia-1). D , cells were cotransfected with GFP and Dia-1/control siRNA, and migration stimulated by CML-HAS or 10% fetal bovine serum control ( FBS ) was performed and quantified as described above. The results are the means ± S.D. ( n = 3).
    Streptavidin Horseradish Peroxidase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher horseradish conjugated streptavidin
    Dia-1 siRNA blocks RAGE ligand-stimulated cellular migration. C6 glioma cells stably expressing RAGE were transfected with siRNAs against Dia-1 or siRNA control. 48 h after transfection cells were lysed, and Dia-1 levels were assessed by quantitative-PCR ( A ) or Western blotting ( WB ) using anti-Dia-1 antibodies ( B ). C , cells were transfected with either Dia-1 siRNA/GFP and scramble siRNA/GFP and, after 48 h, fixed and immunostained with antibodies to Dia-1 followed by respective biotinylated secondary antibodies and <t>streptavidin-linked</t> Alexa-546 (Dia-1). D , cells were cotransfected with GFP and Dia-1/control siRNA, and migration stimulated by CML-HAS or 10% fetal bovine serum control ( FBS ) was performed and quantified as described above. The results are the means ± S.D. ( n = 3).
    Horseradish Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin horseradish conjugate
    Dia-1 siRNA blocks RAGE ligand-stimulated cellular migration. C6 glioma cells stably expressing RAGE were transfected with siRNAs against Dia-1 or siRNA control. 48 h after transfection cells were lysed, and Dia-1 levels were assessed by quantitative-PCR ( A ) or Western blotting ( WB ) using anti-Dia-1 antibodies ( B ). C , cells were transfected with either Dia-1 siRNA/GFP and scramble siRNA/GFP and, after 48 h, fixed and immunostained with antibodies to Dia-1 followed by respective biotinylated secondary antibodies and <t>streptavidin-linked</t> Alexa-546 (Dia-1). D , cells were cotransfected with GFP and Dia-1/control siRNA, and migration stimulated by CML-HAS or 10% fetal bovine serum control ( FBS ) was performed and quantified as described above. The results are the means ± S.D. ( n = 3).
    Streptavidin Horseradish Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase
    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and <t>streptavidin-Alexa555,</t> then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
    Streptavidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish
    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and <t>streptavidin-Alexa555,</t> then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
    Streptavidin Horseradish, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin hrp
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 3079 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies streptavidin horseradish
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Horseradish, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies streptavidin horseradish peroxidiase hrp
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Horseradish Peroxidiase Hrp, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend streptavidin horseradish peroxidase
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Horseradish Peroxidase, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase conjugate
    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on <t>streptavidin</t> magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.
    Streptavidin Horseradish Peroxidase Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 864 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin hrp conjugate
    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 <t>streptavidin-HRP</t> 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.
    Streptavidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 610 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam horseradish peroxidase streptavidin
    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 <t>streptavidin-HRP</t> 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.
    Horseradish Peroxidase Streptavidin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dia-1 siRNA blocks RAGE ligand-stimulated cellular migration. C6 glioma cells stably expressing RAGE were transfected with siRNAs against Dia-1 or siRNA control. 48 h after transfection cells were lysed, and Dia-1 levels were assessed by quantitative-PCR ( A ) or Western blotting ( WB ) using anti-Dia-1 antibodies ( B ). C , cells were transfected with either Dia-1 siRNA/GFP and scramble siRNA/GFP and, after 48 h, fixed and immunostained with antibodies to Dia-1 followed by respective biotinylated secondary antibodies and streptavidin-linked Alexa-546 (Dia-1). D , cells were cotransfected with GFP and Dia-1/control siRNA, and migration stimulated by CML-HAS or 10% fetal bovine serum control ( FBS ) was performed and quantified as described above. The results are the means ± S.D. ( n = 3).

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction of the RAGE Cytoplasmic Domain with Diaphanous-1 Is Required for Ligand-stimulated Cellular Migration through Activation of Rac1 and Cdc42 *Interaction of the RAGE Cytoplasmic Domain with Diaphanous-1 Is Required for Ligand-stimulated Cellular Migration through Activation of Rac1 and Cdc42 * S⃞

    doi: 10.1074/jbc.M801465200

    Figure Lengend Snippet: Dia-1 siRNA blocks RAGE ligand-stimulated cellular migration. C6 glioma cells stably expressing RAGE were transfected with siRNAs against Dia-1 or siRNA control. 48 h after transfection cells were lysed, and Dia-1 levels were assessed by quantitative-PCR ( A ) or Western blotting ( WB ) using anti-Dia-1 antibodies ( B ). C , cells were transfected with either Dia-1 siRNA/GFP and scramble siRNA/GFP and, after 48 h, fixed and immunostained with antibodies to Dia-1 followed by respective biotinylated secondary antibodies and streptavidin-linked Alexa-546 (Dia-1). D , cells were cotransfected with GFP and Dia-1/control siRNA, and migration stimulated by CML-HAS or 10% fetal bovine serum control ( FBS ) was performed and quantified as described above. The results are the means ± S.D. ( n = 3).

    Article Snippet: Western blotting was performed using 4-12% NuPAGE Bis-Tris gel with MOPS buffer (Invitrogen), nitrocellulose membranes (Invitrogen), incubation with anti-HisG antibodies (Invitrogen), or streptavidin-horseradish peroxidase (Promega).

    Techniques: Migration, Stable Transfection, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot

    Diaphanous-1 interacts with the RAGE cytoplasmic domain: in vitro and in vivo studies. A , the specificity of the interaction of Dia-1 with the RAGE cytoplasmic domain was analyzed in vitro by GST pulldown assays. GST only or GST-RAGE cytoplasmic tail fusion proteins were incubated with in vitro transcribed/translated His-tagged Dia-YIIH or His tag only (empty vector) labeled with biotinylated lysine-tRNA complex (Transcend™ tRNA). GST pulldown assays were performed, and eluted proteins were analyzed by Western blotting ( WB ) with streptavidin-horseradish peroxidase ( Strep-HRP ). An equivalent amount of the GST fusion proteins along with a sample from the in vitro transcription/translation reaction were analyzed by Western blotting using anti-His and anti-GST antibodies ( lower panel ). B , the RAGE cytoplasmic domain and Dia-1 interaction was analyzed by coimmunoprecipitation ( IP ) experiments with cell lysates from SK-BR-3 cells transiently transfected with His-tagged RAGE cytoplasmic domain or empty vector and Myc-tagged Dia-1 or empty vector. Cell extracts were immunoprecipitated with anti-His or anti-Myc antibodies and analyzed by Western blotting using anti-His and anti-Myc antibodies. C , C6 glioma cells stably expressing RAGE or the cytoplasmic domain-deleted RAGE ( DN-RAGE ) were analyzed for interaction with endogenous Dia-1 by coimmunoprecipitation experiments. Cells were serum-starved for 24 h followed by stimulation with CML-HSA (10 μg/ml) for 30 min. Cell extracts were then immunoprecipitated with anti-RAGE antibodies and analyzed by Western blotting with anti-Dia-1 antibodies. Total extract used for immunoprecipitation is indicated as input lysate and was analyzed by Western blotting with anti-RAGE and anti-Dia-1 antibodies. All figures ( A-C ) are representative images of multiple independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction of the RAGE Cytoplasmic Domain with Diaphanous-1 Is Required for Ligand-stimulated Cellular Migration through Activation of Rac1 and Cdc42 *Interaction of the RAGE Cytoplasmic Domain with Diaphanous-1 Is Required for Ligand-stimulated Cellular Migration through Activation of Rac1 and Cdc42 * S⃞

    doi: 10.1074/jbc.M801465200

    Figure Lengend Snippet: Diaphanous-1 interacts with the RAGE cytoplasmic domain: in vitro and in vivo studies. A , the specificity of the interaction of Dia-1 with the RAGE cytoplasmic domain was analyzed in vitro by GST pulldown assays. GST only or GST-RAGE cytoplasmic tail fusion proteins were incubated with in vitro transcribed/translated His-tagged Dia-YIIH or His tag only (empty vector) labeled with biotinylated lysine-tRNA complex (Transcend™ tRNA). GST pulldown assays were performed, and eluted proteins were analyzed by Western blotting ( WB ) with streptavidin-horseradish peroxidase ( Strep-HRP ). An equivalent amount of the GST fusion proteins along with a sample from the in vitro transcription/translation reaction were analyzed by Western blotting using anti-His and anti-GST antibodies ( lower panel ). B , the RAGE cytoplasmic domain and Dia-1 interaction was analyzed by coimmunoprecipitation ( IP ) experiments with cell lysates from SK-BR-3 cells transiently transfected with His-tagged RAGE cytoplasmic domain or empty vector and Myc-tagged Dia-1 or empty vector. Cell extracts were immunoprecipitated with anti-His or anti-Myc antibodies and analyzed by Western blotting using anti-His and anti-Myc antibodies. C , C6 glioma cells stably expressing RAGE or the cytoplasmic domain-deleted RAGE ( DN-RAGE ) were analyzed for interaction with endogenous Dia-1 by coimmunoprecipitation experiments. Cells were serum-starved for 24 h followed by stimulation with CML-HSA (10 μg/ml) for 30 min. Cell extracts were then immunoprecipitated with anti-RAGE antibodies and analyzed by Western blotting with anti-Dia-1 antibodies. Total extract used for immunoprecipitation is indicated as input lysate and was analyzed by Western blotting with anti-RAGE and anti-Dia-1 antibodies. All figures ( A-C ) are representative images of multiple independent experiments.

    Article Snippet: Western blotting was performed using 4-12% NuPAGE Bis-Tris gel with MOPS buffer (Invitrogen), nitrocellulose membranes (Invitrogen), incubation with anti-HisG antibodies (Invitrogen), or streptavidin-horseradish peroxidase (Promega).

    Techniques: In Vitro, In Vivo, Incubation, Plasmid Preparation, Labeling, Western Blot, Transfection, Immunoprecipitation, Stable Transfection, Expressing

    RAGE and Dia-1 colocalization in cells. Confocal microscopy was performed using the C6 cells stably transfected with either full-length RAGE ( A ) or empty vector ( Mock ) ( B ) constructs. Cells were treated with RAGE ligand for 30 min, fixed, and immunostained with antibodies to RAGE and Dia-1 followed by respective biotinylated secondary antibodies and streptavidin-linked Alexa-546 ( anti-RAGE ) and 488 ( anti-Dia-1 ). Higher magnification images of the cells within the stippled boxes in the upper panels are illustrated in the lower panels .

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction of the RAGE Cytoplasmic Domain with Diaphanous-1 Is Required for Ligand-stimulated Cellular Migration through Activation of Rac1 and Cdc42 *Interaction of the RAGE Cytoplasmic Domain with Diaphanous-1 Is Required for Ligand-stimulated Cellular Migration through Activation of Rac1 and Cdc42 * S⃞

    doi: 10.1074/jbc.M801465200

    Figure Lengend Snippet: RAGE and Dia-1 colocalization in cells. Confocal microscopy was performed using the C6 cells stably transfected with either full-length RAGE ( A ) or empty vector ( Mock ) ( B ) constructs. Cells were treated with RAGE ligand for 30 min, fixed, and immunostained with antibodies to RAGE and Dia-1 followed by respective biotinylated secondary antibodies and streptavidin-linked Alexa-546 ( anti-RAGE ) and 488 ( anti-Dia-1 ). Higher magnification images of the cells within the stippled boxes in the upper panels are illustrated in the lower panels .

    Article Snippet: Western blotting was performed using 4-12% NuPAGE Bis-Tris gel with MOPS buffer (Invitrogen), nitrocellulose membranes (Invitrogen), incubation with anti-HisG antibodies (Invitrogen), or streptavidin-horseradish peroxidase (Promega).

    Techniques: Confocal Microscopy, Stable Transfection, Transfection, Plasmid Preparation, Construct

    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm

    Journal: BMC Evolutionary Biology

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein

    doi: 10.1186/s12862-016-0652-x

    Figure Lengend Snippet: Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm

    Article Snippet: For GFP detection, membranes were incubated with a rabbit polyclonal antibody (Life Technologies, A-11122) diluted 1:2000 in 50 mM Tris, 100 mM NaCl, 0.1 % Tween 20, pH 7.4, with 3 % bovine serum albumine (Sigma, St. Louis, MO) and a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Waltham, MA), while for HA revelation, they were incubated with biotinylated 3 F10 rat monoclonal antibody (Roche Life Science, Indianapolis, IN) diluted 1:500 in the same buffer, and streptavidin-horseradish peroxidase (Invitrogen, Waltham, MA).

    Techniques: Expressing, Transfection, Staining

    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Journal: BMC Cell Biology

    Article Title: A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells

    doi: 10.1186/s12860-014-0045-1

    Figure Lengend Snippet: Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Article Snippet: Each diluted standard was incubated with primary antibody coated on the well and streptavidin-HRP and OD value for each sample was detected by ELISA.

    Techniques: Labeling, Western Blot, Synthesized, Conjugation Assay, Transfection, Purification, Immunoprecipitation, Autoradiography

    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on streptavidin magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.

    Journal: Analytical Chemistry

    Article Title: Robust, Quantitative Analysis of Proteins using Peptide Immunoreagents, in Vitro Translation, and an Ultrasensitive Acoustic Resonant Sensor

    doi: 10.1021/ac500084d

    Figure Lengend Snippet: AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on streptavidin magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.

    Article Snippet: Plates were washed with wash buffer [1× PBS + 0.1% (v/v) Tween-20] and blocked with 1× PBS + 5% (w/v) BSA for 2 h. In each well, 100 μL of sample and 100 μL of 33 nM biotinylated Bcl-xL were added and incubated for 4 h. Plates were washed, incubated with streptavidin horseradish peroxidase conjugate (Strep-HRP, Thermo Scientific) for 1 h, washed, and incubated with TMB substrate (Thermo Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Magnetic Beads, Labeling, Concentration Assay, Competitive Binding Assay

    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