horseradish peroxidase streptavidin conjugate  (Thermo Fisher)


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    Structured Review

    Thermo Fisher horseradish peroxidase streptavidin conjugate
    Horseradish Peroxidase Streptavidin Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase streptavidin conjugate/product/Thermo Fisher
    Average 80 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase streptavidin conjugate - by Bioz Stars, 2020-03
    80/100 stars

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    Related Articles

    Amplification:

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour. .. Finally, prior to treatment with tyramide solution for the signal amplification of the target mRNA transcripts, samples were washed twice with PBT and once with DPBS.

    Blocking Assay:

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: Next, to reduce non-specific staining, samples were blocked with PBTB (DPBS, 0.1% Tween-20, 1% Molecular Probes block reagent; Invitrogen, Carlsbad, CA) for one hour. .. Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Angiogenesis blockade as a new therapeutic approach to experimental colitis
    Article Snippet: Briefly, 96‐well ELISA plates (Dynatech, Chantilly, Virginia, USA) were coated for 1 h at 37°C with 4 μg/ml of C15.6 monoclonal antibody against mouse IL12 (grown as an ascites fluid in nude mice and purified by protein G Sepharose; Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). .. After washing five times, a 1:4000 dilution of a horseradish peroxidase–streptavidin conjugate (Zymed, San Francisco, California, USA) was added to each well for 30 min at room temperature.

    Article Title: Serum thymus and activation regulated chemokine levels post-lung transplantation as a predictor for the bronchiolitis obliterans syndrome
    Article Snippet: Paragraph title: Enzyme-linked immunosorbent assay ... Horseradish peroxidase–streptavidin conjugate (Zymed, San Francisco, CA, USA) and substrate [3.3′, 5.5′-tetramethylbenzidine substrate; Pierce, Rockford, IL, USA] were used according to the manufacturer's manual.

    Article Title: Basophils Initiate IL-4 Production during a Memory T-dependent Response
    Article Snippet: .. Cytokine-biotin–anti-cytokine mAb complexes accumulate to much higher levels than free cytokines in serum and are measured by ELISA, using microtiter plate wells coated with mAb to a second, noncompeting epitope on the cytokine molecule to capture the complex and a horseradish peroxidase–streptavidin conjugate (Pierce Chemical Co.) and a luminogenic substrate for horseradish peroxidase (SuperSignal ELISA femto-substrate; Pierce Chemical Co.) to detect the captured complex. ..

    Sandwich ELISA:

    Article Title: Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4
    Article Snippet: Paragraph title: Sandwich ELISA. ... Horseradish peroxidase-streptavidin conjugate was added (1:4,000; Zymed, San Francisco, Calif.) with 3,3,5,5-tetramethylbenzidine (Zymed) substrate.

    Incubation:

    Article Title: Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4
    Article Snippet: Wells were washed, and 100 μl of biotinylated anti-human KGF-1 polyclonal antibody (BAF251; 200 ng/ml; R & D Systems Inc.) was added, the wells were incubated for 2 h, and then the wells were washed. .. Horseradish peroxidase-streptavidin conjugate was added (1:4,000; Zymed, San Francisco, Calif.) with 3,3,5,5-tetramethylbenzidine (Zymed) substrate.

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: .. Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour. .. Finally, prior to treatment with tyramide solution for the signal amplification of the target mRNA transcripts, samples were washed twice with PBT and once with DPBS.

    Article Title: Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect
    Article Snippet: .. Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C. .. Labeling was visualized with chromogen diaminobenzidine (no. 00-2014, Zymed Laboratories).

    Proliferation Assay:

    Article Title: Angiogenesis blockade as a new therapeutic approach to experimental colitis
    Article Snippet: Paragraph title: Measurement of mucosal and cell‐derived cytokines and proliferation assay ... After washing five times, a 1:4000 dilution of a horseradish peroxidase–streptavidin conjugate (Zymed, San Francisco, California, USA) was added to each well for 30 min at room temperature.

    Hybridization:

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: The following day, samples were washed twice with fresh hybridization solution (minus probe) and subsequently with 3:1, 1:1 and 1:3 (vol/vol) mixtures of hybridization solution-PBT (all pre-warmed to 56 °C). .. Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour.

    Immunohistochemistry:

    Article Title: Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect
    Article Snippet: Paragraph title: Immunohistochemistry. ... Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C.

    Injection:

    Article Title: Basophils Initiate IL-4 Production during a Memory T-dependent Response
    Article Snippet: Mice are injected with 10 μg of biotin-labeled, neutralizing anticytokine mAb, which binds secreted cytokine. .. Cytokine-biotin–anti-cytokine mAb complexes accumulate to much higher levels than free cytokines in serum and are measured by ELISA, using microtiter plate wells coated with mAb to a second, noncompeting epitope on the cytokine molecule to capture the complex and a horseradish peroxidase–streptavidin conjugate (Pierce Chemical Co.) and a luminogenic substrate for horseradish peroxidase (SuperSignal ELISA femto-substrate; Pierce Chemical Co.) to detect the captured complex.

    Recombinant:

    Article Title: Angiogenesis blockade as a new therapeutic approach to experimental colitis
    Article Snippet: After five washes with PBS/Tween, 40 μl of supernatant or murine recombinant IL12 standard (5000–78 pg/ml in twofold serial dilutions; R & D Systems, Minneapolis, Minnesota, USA) was added to the plate in duplicate at room temperature for 2 h. The plate was washed five times with PBS/Tween, followed by 1 μg/ml of biotinylated C17.8 (PharMingen, San Diego, California, USA) for 1 h at room temperature. .. After washing five times, a 1:4000 dilution of a horseradish peroxidase–streptavidin conjugate (Zymed, San Francisco, California, USA) was added to each well for 30 min at room temperature.

    Article Title: Serum thymus and activation regulated chemokine levels post-lung transplantation as a predictor for the bronchiolitis obliterans syndrome
    Article Snippet: Human serum diluted (1/2) was added and standard concentrations (range: 4000 pg/ml–16 pg/ml) were prepared with recombinant human TARC (364-DN; R & D Systems) in phosphate-buffered saline containing 1% bovine serum albumin. .. Horseradish peroxidase–streptavidin conjugate (Zymed, San Francisco, CA, USA) and substrate [3.3′, 5.5′-tetramethylbenzidine substrate; Pierce, Rockford, IL, USA] were used according to the manufacturer's manual.

    In Vivo:

    Article Title: Basophils Initiate IL-4 Production during a Memory T-dependent Response
    Article Snippet: Paragraph title: In Vivo Cytokine Capture Assay (IVCCA). ... Cytokine-biotin–anti-cytokine mAb complexes accumulate to much higher levels than free cytokines in serum and are measured by ELISA, using microtiter plate wells coated with mAb to a second, noncompeting epitope on the cytokine molecule to capture the complex and a horseradish peroxidase–streptavidin conjugate (Pierce Chemical Co.) and a luminogenic substrate for horseradish peroxidase (SuperSignal ELISA femto-substrate; Pierce Chemical Co.) to detect the captured complex.

    Fluorescence:

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: Paragraph title: Fluorescence in situ Hybridization (FISH) ... Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour.

    Labeling:

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour. .. After experimenting with various dilutions of the labeled tyramide, a 1:100 and 1:500 dilution of tyramide dye gave optimal results with minimal background staining for the ganglia and MTs, respectively.

    Article Title: Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect
    Article Snippet: Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C. .. Labeling was visualized with chromogen diaminobenzidine (no. 00-2014, Zymed Laboratories).

    Purification:

    Article Title: Angiogenesis blockade as a new therapeutic approach to experimental colitis
    Article Snippet: Briefly, 96‐well ELISA plates (Dynatech, Chantilly, Virginia, USA) were coated for 1 h at 37°C with 4 μg/ml of C15.6 monoclonal antibody against mouse IL12 (grown as an ascites fluid in nude mice and purified by protein G Sepharose; Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). .. After washing five times, a 1:4000 dilution of a horseradish peroxidase–streptavidin conjugate (Zymed, San Francisco, California, USA) was added to each well for 30 min at room temperature.

    Mouse Assay:

    Article Title: Angiogenesis blockade as a new therapeutic approach to experimental colitis
    Article Snippet: Briefly, 96‐well ELISA plates (Dynatech, Chantilly, Virginia, USA) were coated for 1 h at 37°C with 4 μg/ml of C15.6 monoclonal antibody against mouse IL12 (grown as an ascites fluid in nude mice and purified by protein G Sepharose; Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). .. After washing five times, a 1:4000 dilution of a horseradish peroxidase–streptavidin conjugate (Zymed, San Francisco, California, USA) was added to each well for 30 min at room temperature.

    Article Title: Basophils Initiate IL-4 Production during a Memory T-dependent Response
    Article Snippet: Mice are injected with 10 μg of biotin-labeled, neutralizing anticytokine mAb, which binds secreted cytokine. .. Cytokine-biotin–anti-cytokine mAb complexes accumulate to much higher levels than free cytokines in serum and are measured by ELISA, using microtiter plate wells coated with mAb to a second, noncompeting epitope on the cytokine molecule to capture the complex and a horseradish peroxidase–streptavidin conjugate (Pierce Chemical Co.) and a luminogenic substrate for horseradish peroxidase (SuperSignal ELISA femto-substrate; Pierce Chemical Co.) to detect the captured complex.

    Article Title: Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect
    Article Snippet: The kidneys of 7-day dDAVP-infused AQP1KO mice were fixed in 4% paraformaldehyde overnight at 4°C. .. Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C.

    In Situ Hybridization:

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: Paragraph title: Fluorescence in situ Hybridization (FISH) ... Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour.

    Plasmid Preparation:

    Article Title: Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect
    Article Snippet: Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C. .. Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C.

    Binding Assay:

    Article Title: Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect
    Article Snippet: Endogenous peroxidase activity was quenched in absolute methanol plus 0.3% (vol/vol) hydrogen peroxide for 30 min. Antigen was retrieved by heating the sections in citrate buffer (pH 6.0) in a microwave oven for 10 min. Nonspecific binding was blocked in 2% BSA. .. Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C.

    Concentration Assay:

    Article Title: Serum thymus and activation regulated chemokine levels post-lung transplantation as a predictor for the bronchiolitis obliterans syndrome
    Article Snippet: Horseradish peroxidase–streptavidin conjugate (Zymed, San Francisco, CA, USA) and substrate [3.3′, 5.5′-tetramethylbenzidine substrate; Pierce, Rockford, IL, USA] were used according to the manufacturer's manual. .. The minimal concentration of TARC that could be detected was 16 pg/ml.

    Staining:

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: Next, to reduce non-specific staining, samples were blocked with PBTB (DPBS, 0.1% Tween-20, 1% Molecular Probes block reagent; Invitrogen, Carlsbad, CA) for one hour. .. Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour.

    Article Title: Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect
    Article Snippet: Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C. .. For immunofluorescent staining, the protocol used was the same as that for immunohistochemistry except for the secondary antibodies, which were FITC-conjugated donkey anti-rabbit (1:200 dilution, no. 711-095-12, Jackson ImmunoResearch, West Grove, PA) and Texas Red conjugated donkey anti-goat (1:200 dilution, no. 705-075-147, Jackson ImmunoResearch,).

    Fluorescence In Situ Hybridization:

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti
    Article Snippet: Paragraph title: Fluorescence in situ Hybridization (FISH) ... Tissues/organs were then incubated with horseradish peroxidase-streptavidin conjugate (Molecular Probes, Eugene, OR) diluted 1:100 in PBTB for 1 hour and the tissues were once again washed with PBTB several times over the course of an hour.

    Activity Assay:

    Article Title: Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect
    Article Snippet: Endogenous peroxidase activity was quenched in absolute methanol plus 0.3% (vol/vol) hydrogen peroxide for 30 min. Antigen was retrieved by heating the sections in citrate buffer (pH 6.0) in a microwave oven for 10 min. Nonspecific binding was blocked in 2% BSA. .. Sections were then incubated overnight at 4°C with rabbit anti-GRP78, followed by biotin-conjugated goat anti-rabbit IgG (1:200 dilution, no. 81-6140, Zymed Laboratories, South San Francisco, CA) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution, no. 43-4323, Zymed Laboratories) for 30 min at 37°C.

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    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase conjugated streptavidin/product/Thermo Fisher
    Average 99 stars, based on 90 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase conjugated streptavidin - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher streptavidin conjugated hrp
    Photoaffinity labeling of various PrP species. <t>Streptavidin-HRP-probed</t> blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.
    Streptavidin Conjugated Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated hrp/product/Thermo Fisher
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    streptavidin conjugated hrp - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

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

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

    Journal: Journal of Proteome Research

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

    doi: 10.1021/pr200918f

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

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

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

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

    Journal: Biochemistry

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

    doi: 10.1021/bi4014825

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

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

    Techniques: Labeling, Incubation

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms7765

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

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

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