proteinase inhibitor cocktail  (Millipore)

 
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    Name:
    Protease Inhibitor Cocktail
    Description:

    Catalog Number:
    p8340
    Price:
    None
    Applications:
    Protease Inhibitor Cocktail has been used-. as a buffer component during sonication of GFP (green fluorescent protein)-huntingtin-transfected HEK 293 cells in GST (glutathione S-transferase) pull down assay. as a component of lysis buffer. as a component of radioimmunoprecipitation assay buffer (RIPA)
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    Structured Review

    Millipore proteinase inhibitor cocktail

    https://www.bioz.com/result/proteinase inhibitor cocktail/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase inhibitor cocktail - by Bioz Stars, 2021-03
    86/100 stars

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    Related Articles

    Protease Inhibitor:

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells
    Article Snippet: Lovastatin (Enzo Life Sciences Inc., Farmingdale, NY) was activated by the addition of 0,1 N NaOH (lovastatin/NaOH 2:3 v/v), at 50°C and neutralized with 1 N HCl to pH 7,2. .. Phenylmethylsulfonylfuoride (PMSF) (Euroclone, Devon, UK), pepstatin A (Boehringer, Mannheim, DE), GM6001 inhibitor (Biomol Research Lab Inc.), protease inhibitor cocktail set III (AEBSF 100mM; aprotinin 80μM; bestatin 5mM; E-64 1,5mM; leupeptin 2mM; pepstatin A 1mM) (Calbiochem, La Jolla, CA) were used for specific protease class inhibition experiments. .. Ox-LDL preparation, labelling and fluorescence analysis Human LDL was prepared from fresh healthy normolipidemic plasma of volunteers by ultracentrifugation [ ] and in vitro oxidized as described (Matarazzo et al., 2012).

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
    Article Snippet: HEK293 cells cultured in a 12-well plate were transfected with plasmids encoding pEGFP, pEGFP-RAB5 (WT, DN, or CA), pcDNA3.1-MYC-SGSM3 (RABGAP5), or pcDNA3.1-MYC-SGSM3R165A , and infected with E. chaffeensis at 1 d p.t. for 2 d. Cells were lysed in 1× SDS sample buffer, sonicated, and examined by western blotting using antibodies against EGFP (for RAB5), MYC/c-Myc (for SGSM3), and E. chaffeensis P28. .. Co-immunoprecipitation Uninfected or E. chaffeensis-infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805). .. Normal rabbit (Santa Cruz Biotechnology, sc-2027) or mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as a negative control for immunoprecipitation.

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Article Title: Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace
    Article Snippet: Cells were blocked with anti-CD16/CD32 (BD Biosciences, San Jose, CA) for 20 min on ice and processed for flow cytometry. .. For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C. .. Protein lysate samples were used for mouse neutrophil elastase/ELA2 and MPO ELISA (R & D Systems, MN) per manufacturers protocol.

    Article Title: Nasal Immunity to Staphylococcal Toxic Shock Is Controlled by the Nasopharynx-Associated Lymphoid Tissue ▿Nasal Immunity to Staphylococcal Toxic Shock Is Controlled by the Nasopharynx-Associated Lymphoid Tissue ▿ †Nasal Immunity to Staphylococcal Toxic Shock Is Controlled by the Nasopharynx-Associated Lymphoid Tissue ▿ † ‡
    Article Snippet: .. The collected rinse (5 to 8 μl) was deposited in a fresh centrifuge tube containing 10 μl PBS with 2× protease inhibitor cocktail set III (Calbiochem, San Diego, CA). .. Nasal lavage was collected from euthanized (CO2 asphyxiation, followed by cervical dislocation) mice by carefully introducing 20 to 30 μl PBS with a pipette through one nostril.

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: Samples were acquired on a BD LSR II (BD Biosciences) and analysed using FlowJo software (Treestar, Inc., Ashland, OR, USA). .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

    Inhibition:

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells
    Article Snippet: Lovastatin (Enzo Life Sciences Inc., Farmingdale, NY) was activated by the addition of 0,1 N NaOH (lovastatin/NaOH 2:3 v/v), at 50°C and neutralized with 1 N HCl to pH 7,2. .. Phenylmethylsulfonylfuoride (PMSF) (Euroclone, Devon, UK), pepstatin A (Boehringer, Mannheim, DE), GM6001 inhibitor (Biomol Research Lab Inc.), protease inhibitor cocktail set III (AEBSF 100mM; aprotinin 80μM; bestatin 5mM; E-64 1,5mM; leupeptin 2mM; pepstatin A 1mM) (Calbiochem, La Jolla, CA) were used for specific protease class inhibition experiments. .. Ox-LDL preparation, labelling and fluorescence analysis Human LDL was prepared from fresh healthy normolipidemic plasma of volunteers by ultracentrifugation [ ] and in vitro oxidized as described (Matarazzo et al., 2012).

    Modification:

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
    Article Snippet: HEK293 cells cultured in a 12-well plate were transfected with plasmids encoding pEGFP, pEGFP-RAB5 (WT, DN, or CA), pcDNA3.1-MYC-SGSM3 (RABGAP5), or pcDNA3.1-MYC-SGSM3R165A , and infected with E. chaffeensis at 1 d p.t. for 2 d. Cells were lysed in 1× SDS sample buffer, sonicated, and examined by western blotting using antibodies against EGFP (for RAB5), MYC/c-Myc (for SGSM3), and E. chaffeensis P28. .. Co-immunoprecipitation Uninfected or E. chaffeensis-infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805). .. Normal rabbit (Santa Cruz Biotechnology, sc-2027) or mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as a negative control for immunoprecipitation.

    Lysis:

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
    Article Snippet: HEK293 cells cultured in a 12-well plate were transfected with plasmids encoding pEGFP, pEGFP-RAB5 (WT, DN, or CA), pcDNA3.1-MYC-SGSM3 (RABGAP5), or pcDNA3.1-MYC-SGSM3R165A , and infected with E. chaffeensis at 1 d p.t. for 2 d. Cells were lysed in 1× SDS sample buffer, sonicated, and examined by western blotting using antibodies against EGFP (for RAB5), MYC/c-Myc (for SGSM3), and E. chaffeensis P28. .. Co-immunoprecipitation Uninfected or E. chaffeensis-infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805). .. Normal rabbit (Santa Cruz Biotechnology, sc-2027) or mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as a negative control for immunoprecipitation.

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: Samples were acquired on a BD LSR II (BD Biosciences) and analysed using FlowJo software (Treestar, Inc., Ashland, OR, USA). .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

    Immunoprecipitation:

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
    Article Snippet: HEK293 cells cultured in a 12-well plate were transfected with plasmids encoding pEGFP, pEGFP-RAB5 (WT, DN, or CA), pcDNA3.1-MYC-SGSM3 (RABGAP5), or pcDNA3.1-MYC-SGSM3R165A , and infected with E. chaffeensis at 1 d p.t. for 2 d. Cells were lysed in 1× SDS sample buffer, sonicated, and examined by western blotting using antibodies against EGFP (for RAB5), MYC/c-Myc (for SGSM3), and E. chaffeensis P28. .. Co-immunoprecipitation Uninfected or E. chaffeensis-infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805). .. Normal rabbit (Santa Cruz Biotechnology, sc-2027) or mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as a negative control for immunoprecipitation.

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: Samples were acquired on a BD LSR II (BD Biosciences) and analysed using FlowJo software (Treestar, Inc., Ashland, OR, USA). .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Recombinant:

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Western Blot:

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Fractionation:

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: Samples were acquired on a BD LSR II (BD Biosciences) and analysed using FlowJo software (Treestar, Inc., Ashland, OR, USA). .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Cell Culture:

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: Samples were acquired on a BD LSR II (BD Biosciences) and analysed using FlowJo software (Treestar, Inc., Ashland, OR, USA). .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Incubation:

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

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  • 93
    Millipore caspase assay buffer
    Depletion of Wip1 enhances IR-induced apoptotic cell death. ( a ) HeLa or LNCaP cells were irradiated with 10 Gy. After 72 h, the cells were harvested and stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). The proportions of apoptotic cells within the annexin V-FITC and PI-positive subpopulations were determined using fluorescence-activated cell sorting (FACS). ( b ) LNCaP cells were transfected with 50 nM of either control small interfering (si)RNA or Wip1 siRNA. At 48 h post-transfection, the cells were irradiated with 10 Gy. After 72 h, the cells were stained with PI and annexin V-FITC. Apoptotic cell death was determined by FACS. ( c ) LNCaP cells were transfected with 50 nM of either control siRNA or Wip1 siRNA for 36 h, and then irradiated with 10 Gy. After 40 h, the transfectants were assayed for <t>caspase-3</t> activity. All assays were performed in triplicate. The data show the mean±S.D. (* P
    Caspase Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase assay buffer/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    caspase assay buffer - by Bioz Stars, 2021-03
    93/100 stars
      Buy from Supplier

    97
    Millipore proteinase inhibitors
    Depletion of Wip1 enhances IR-induced apoptotic cell death. ( a ) HeLa or LNCaP cells were irradiated with 10 Gy. After 72 h, the cells were harvested and stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). The proportions of apoptotic cells within the annexin V-FITC and PI-positive subpopulations were determined using fluorescence-activated cell sorting (FACS). ( b ) LNCaP cells were transfected with 50 nM of either control small interfering (si)RNA or Wip1 siRNA. At 48 h post-transfection, the cells were irradiated with 10 Gy. After 72 h, the cells were stained with PI and annexin V-FITC. Apoptotic cell death was determined by FACS. ( c ) LNCaP cells were transfected with 50 nM of either control siRNA or Wip1 siRNA for 36 h, and then irradiated with 10 Gy. After 40 h, the transfectants were assayed for <t>caspase-3</t> activity. All assays were performed in triplicate. The data show the mean±S.D. (* P
    Proteinase Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase inhibitors/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase inhibitors - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier

    97
    Millipore proteinase inhibitor cocktail
    Depletion of Wip1 enhances IR-induced apoptotic cell death. ( a ) HeLa or LNCaP cells were irradiated with 10 Gy. After 72 h, the cells were harvested and stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). The proportions of apoptotic cells within the annexin V-FITC and PI-positive subpopulations were determined using fluorescence-activated cell sorting (FACS). ( b ) LNCaP cells were transfected with 50 nM of either control small interfering (si)RNA or Wip1 siRNA. At 48 h post-transfection, the cells were irradiated with 10 Gy. After 72 h, the cells were stained with PI and annexin V-FITC. Apoptotic cell death was determined by FACS. ( c ) LNCaP cells were transfected with 50 nM of either control siRNA or Wip1 siRNA for 36 h, and then irradiated with 10 Gy. After 40 h, the transfectants were assayed for <t>caspase-3</t> activity. All assays were performed in triplicate. The data show the mean±S.D. (* P
    Proteinase Inhibitor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase inhibitor cocktail/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase inhibitor cocktail - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier

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    Depletion of Wip1 enhances IR-induced apoptotic cell death. ( a ) HeLa or LNCaP cells were irradiated with 10 Gy. After 72 h, the cells were harvested and stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). The proportions of apoptotic cells within the annexin V-FITC and PI-positive subpopulations were determined using fluorescence-activated cell sorting (FACS). ( b ) LNCaP cells were transfected with 50 nM of either control small interfering (si)RNA or Wip1 siRNA. At 48 h post-transfection, the cells were irradiated with 10 Gy. After 72 h, the cells were stained with PI and annexin V-FITC. Apoptotic cell death was determined by FACS. ( c ) LNCaP cells were transfected with 50 nM of either control siRNA or Wip1 siRNA for 36 h, and then irradiated with 10 Gy. After 40 h, the transfectants were assayed for caspase-3 activity. All assays were performed in triplicate. The data show the mean±S.D. (* P

    Journal: Cell Death & Disease

    Article Title: Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation

    doi: 10.1038/cddis.2013.252

    Figure Lengend Snippet: Depletion of Wip1 enhances IR-induced apoptotic cell death. ( a ) HeLa or LNCaP cells were irradiated with 10 Gy. After 72 h, the cells were harvested and stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). The proportions of apoptotic cells within the annexin V-FITC and PI-positive subpopulations were determined using fluorescence-activated cell sorting (FACS). ( b ) LNCaP cells were transfected with 50 nM of either control small interfering (si)RNA or Wip1 siRNA. At 48 h post-transfection, the cells were irradiated with 10 Gy. After 72 h, the cells were stained with PI and annexin V-FITC. Apoptotic cell death was determined by FACS. ( c ) LNCaP cells were transfected with 50 nM of either control siRNA or Wip1 siRNA for 36 h, and then irradiated with 10 Gy. After 40 h, the transfectants were assayed for caspase-3 activity. All assays were performed in triplicate. The data show the mean±S.D. (* P

    Article Snippet: Aliquots of protein (50 μ g) were diluted to 50 μ l with Triton X-100 lysis buffer and then incubated for 1 h with an equal volume of 2 × caspase assay buffer (100 mM HEPES, 10% sucrose, 0.1% CHAPS, 1 mM PMSF, 1 mM DTT, 1 × proteinase inhibitor cocktail) containing 50 μ M of fluorogenic (Ac-DEVD-AMC) caspase-3 substrate (235425; Calbiochem, Billerica, MA, USA).

    Techniques: Irradiation, Staining, Fluorescence, FACS, Transfection, Activity Assay

    Wip1 dephosphorylates active BAX and suppresses mitochondria-dependent apoptosis. ( a ) Recombinant Wip1 does not dephosphorylate BAX-derived phosphopeptides encompassing Ser87, Ser163, Ser 184, and Thr167, as shown in an in vitro phosphatase assay. Released free phosphate was measured by absorbance at 630 nm in the presence of molybdate dye. Phosphopeptides p180T from p38 MAPK and p31T from UNG2 were used as positive and negative controls, respectively. 10 ( b ) Phosphorylation sites on Ser and Thr residues in BAX were predicted with the NetPhos 2.0 server. 30 ( c ) Expression vectors carrying GFP wild-type or one of nine mutated GFP-BAX mutant constructs were transfected into HeLa cells. At 48 h post-transfection, the cells were exposed to γ -radiation and then analyzed 24 h later. BAX translocation to the mitochondria results in intense green fluorescence (phosphothreonine and phosphoserine are indicated in bold). ( d ) BAX p172T, p174T, and p186T phosphopeptides were incubated with recombinant Wip1 protein. Free phosphate released by dephosphorylation was measured. Two polypeptides of p38 MAPK p180T served as the positive control, and UNG2 p31T as the negative control. 10 Assays were also performed in the absence of magnesium or Wip1 and in the presence of okadaic acid. Wip1 phosphatase activity is magnesium-dependent and insensitive to okadaic acid. ( e ) DU145 cells were transfected with BAX or mutated BAX (T172D, T174D, and T186D) expression plasmids. After 48 h, the transfectants were irradiated, and then harvested, and finally assayed for caspase-3 activity. ( f ) DU145 cells were transfected with wild-type or mutated BAX together with wild-type Wip1 or phosphatase-dead (PD) Wip1 (D314A and D105A) expression vectors for 48 h and then irradiated with 10 Gy. After 24 h, the transfectants were assayed for caspase-3 activity. The mean of three experiments is shown in each column; bars in ( d ), ( e ), and ( f ) correspond to the S.D. Asterisks (*) indicate P

    Journal: Cell Death & Disease

    Article Title: Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation

    doi: 10.1038/cddis.2013.252

    Figure Lengend Snippet: Wip1 dephosphorylates active BAX and suppresses mitochondria-dependent apoptosis. ( a ) Recombinant Wip1 does not dephosphorylate BAX-derived phosphopeptides encompassing Ser87, Ser163, Ser 184, and Thr167, as shown in an in vitro phosphatase assay. Released free phosphate was measured by absorbance at 630 nm in the presence of molybdate dye. Phosphopeptides p180T from p38 MAPK and p31T from UNG2 were used as positive and negative controls, respectively. 10 ( b ) Phosphorylation sites on Ser and Thr residues in BAX were predicted with the NetPhos 2.0 server. 30 ( c ) Expression vectors carrying GFP wild-type or one of nine mutated GFP-BAX mutant constructs were transfected into HeLa cells. At 48 h post-transfection, the cells were exposed to γ -radiation and then analyzed 24 h later. BAX translocation to the mitochondria results in intense green fluorescence (phosphothreonine and phosphoserine are indicated in bold). ( d ) BAX p172T, p174T, and p186T phosphopeptides were incubated with recombinant Wip1 protein. Free phosphate released by dephosphorylation was measured. Two polypeptides of p38 MAPK p180T served as the positive control, and UNG2 p31T as the negative control. 10 Assays were also performed in the absence of magnesium or Wip1 and in the presence of okadaic acid. Wip1 phosphatase activity is magnesium-dependent and insensitive to okadaic acid. ( e ) DU145 cells were transfected with BAX or mutated BAX (T172D, T174D, and T186D) expression plasmids. After 48 h, the transfectants were irradiated, and then harvested, and finally assayed for caspase-3 activity. ( f ) DU145 cells were transfected with wild-type or mutated BAX together with wild-type Wip1 or phosphatase-dead (PD) Wip1 (D314A and D105A) expression vectors for 48 h and then irradiated with 10 Gy. After 24 h, the transfectants were assayed for caspase-3 activity. The mean of three experiments is shown in each column; bars in ( d ), ( e ), and ( f ) correspond to the S.D. Asterisks (*) indicate P

    Article Snippet: Aliquots of protein (50 μ g) were diluted to 50 μ l with Triton X-100 lysis buffer and then incubated for 1 h with an equal volume of 2 × caspase assay buffer (100 mM HEPES, 10% sucrose, 0.1% CHAPS, 1 mM PMSF, 1 mM DTT, 1 × proteinase inhibitor cocktail) containing 50 μ M of fluorogenic (Ac-DEVD-AMC) caspase-3 substrate (235425; Calbiochem, Billerica, MA, USA).

    Techniques: Recombinant, Derivative Assay, In Vitro, Phosphatase Assay, Expressing, Mutagenesis, Construct, Transfection, Translocation Assay, Fluorescence, Incubation, De-Phosphorylation Assay, Positive Control, Negative Control, Activity Assay, Irradiation