protease inhibitor mixture  (Millipore)


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  • 99
    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 protease inhibitor mixture

    https://www.bioz.com/result/protease inhibitor mixture/product/Millipore
    Average 99 stars, based on 2971 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor mixture - by Bioz Stars, 2020-09
    99/100 stars

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    Protease Inhibitor:

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin
    Article Snippet: .. After 24 h, the medium was replaced with Optimem supplemented with 0.1% protease inhibitor cocktail (PIC) (P8340, Sigma-Aldrich, Saint Louis, MO), 0.1% DMSO, 0.1 mM AEBSF, 0.08 μM aprotinin, 1.4 μM E64, 4 μM bestatin, 2 μM leupeptin and 1.5 μM pepstatin A (Sigma-Aldrich). .. Immunofluorescence analysis was performed after 24 hr of treatment.

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells
    Article Snippet: .. 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: .. 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: Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace
    Article Snippet: .. 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: .. 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).

    Infection:

    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: .. 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.

    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: .. 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: .. 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).

    Inhibition:

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells
    Article Snippet: .. 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).

    Fractionation:

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: .. 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: .. 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. ..

    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: .. 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: .. 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: .. 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).

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  • 99
    Millipore aprotinin
    a , The presence of the three forms of neurocan is not an artifact of storage, concentration, or chondroitinase treatment. The same three bands are seen whether conditioned medium is analyzed before or after storing, concentrating, or chondroitinase treatment. b , Protease inhibitors do not affect neurocan processing. Astrocytes were grown in the presence of <t>aprotinin</t> (1.0 μg/ml), anti-thrombin III (0.1 inhibitor U/ml), or TIMP-2 (0.2 μg/ml) for 4 d. Each inhibitor was added fresh each day. Conditioned medium was concentrated and treated with chondroitinase ABC, and an equal amount of total protein (150 μg) was applied to each lane. The blot was labeled with the 1G2 mAb ( left ) and then relabeled with the 1F6 mAb ( right ). Neither serine protease (aprotinin and anti-thrombin III) nor metalloproteinase (TIMP-2) inhibitors prevented the processing of neurocan.
    Aprotinin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aprotinin/product/Millipore
    Average 99 stars, based on 178 article reviews
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    99
    Millipore z vad fmk
    ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM <t>z-VAD-fmk</t> or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.
    Z Vad Fmk, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/z vad fmk/product/Millipore
    Average 99 stars, based on 187 article reviews
    Price from $9.99 to $1999.99
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    99
    Millipore g418
    The stable expression of V5-tagged rPLD1 in Rat-2 fibroblasts reduces PLD activity. Four <t>G418-resistant</t> clones were selected. (A) The expression of C-terminal V5-tagged rPLD1 was visualized by Western blotting using anti-V5 antibody or an antibody to the C terminus of rPLD1. Lane 1, not Rat-2; lane 2, Rat2V16; lane 3, Rat2V20; lane 4, Rat2V25; lane 5, Rat2V29. The Western blot of endogenous rPLD1 is shown in the lower panel. (B) PLD activity was measured in three clones of Rat2V16, Rat2V25, and Rat2V29 expressing rPLD1-V5 with or without treatment with 10 μg of LPA per ml or 100 nM PMA for 5 or 15 min, respectively. Data are representative of three independent experiments. (C) PLD activity was measured in the Rat2V20 clone, which does not express rPLD1-V5, incubated with or without 10 μg of LPA per ml or 100 nM PMA. Data are representative of two experiments performed in duplicate. Ctrl, control; wt, wild type.
    G418, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a , The presence of the three forms of neurocan is not an artifact of storage, concentration, or chondroitinase treatment. The same three bands are seen whether conditioned medium is analyzed before or after storing, concentrating, or chondroitinase treatment. b , Protease inhibitors do not affect neurocan processing. Astrocytes were grown in the presence of aprotinin (1.0 μg/ml), anti-thrombin III (0.1 inhibitor U/ml), or TIMP-2 (0.2 μg/ml) for 4 d. Each inhibitor was added fresh each day. Conditioned medium was concentrated and treated with chondroitinase ABC, and an equal amount of total protein (150 μg) was applied to each lane. The blot was labeled with the 1G2 mAb ( left ) and then relabeled with the 1F6 mAb ( right ). Neither serine protease (aprotinin and anti-thrombin III) nor metalloproteinase (TIMP-2) inhibitors prevented the processing of neurocan.

    Journal: The Journal of Neuroscience

    Article Title: Neurocan Is Upregulated in Injured Brain and in Cytokine-Treated Astrocytes

    doi: 10.1523/JNEUROSCI.20-07-02427.2000

    Figure Lengend Snippet: a , The presence of the three forms of neurocan is not an artifact of storage, concentration, or chondroitinase treatment. The same three bands are seen whether conditioned medium is analyzed before or after storing, concentrating, or chondroitinase treatment. b , Protease inhibitors do not affect neurocan processing. Astrocytes were grown in the presence of aprotinin (1.0 μg/ml), anti-thrombin III (0.1 inhibitor U/ml), or TIMP-2 (0.2 μg/ml) for 4 d. Each inhibitor was added fresh each day. Conditioned medium was concentrated and treated with chondroitinase ABC, and an equal amount of total protein (150 μg) was applied to each lane. The blot was labeled with the 1G2 mAb ( left ) and then relabeled with the 1F6 mAb ( right ). Neither serine protease (aprotinin and anti-thrombin III) nor metalloproteinase (TIMP-2) inhibitors prevented the processing of neurocan.

    Article Snippet: To investigate neurocan processing, astrocytes were maintained for 4 d in serum-free DMEM containing one of the following protease inhibitors: anti-thrombin III (0.1 U/ml), aprotinin (1.0 μg/ml), tissue inhibitor of metalloproteinase-2 (TIMP-2, 0.2 μg/ml), leupeptin (1.0 μg/ml), E-64 (1.0 μg/ml; Calbiochem, Nottingham, Notts, UK), cathepsin B inhibitor II (1.0 μg/ml; Calbiochem), or α2 -macroglobulin (10 μg/ml).

    Techniques: Concentration Assay, Labeling

    ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM z-VAD-fmk or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.

    Journal: Scientific Reports

    Article Title: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

    doi: 10.1038/s41598-018-20541-8

    Figure Lengend Snippet: ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM z-VAD-fmk or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.

    Article Snippet: JC-1, antibiotic, AlexaFluor 488 and 568 conjugated antibodies, Lipofectamine 2000 were purchased from Invitrogen; z-VAD-fmk, protease inhibitor cocktail and GAPDH antibody were from Millipore.

    Techniques: Transfection, Activity Assay, Staining, Flow Cytometry, Cytometry, Sulforhodamine B Assay

    The stable expression of V5-tagged rPLD1 in Rat-2 fibroblasts reduces PLD activity. Four G418-resistant clones were selected. (A) The expression of C-terminal V5-tagged rPLD1 was visualized by Western blotting using anti-V5 antibody or an antibody to the C terminus of rPLD1. Lane 1, not Rat-2; lane 2, Rat2V16; lane 3, Rat2V20; lane 4, Rat2V25; lane 5, Rat2V29. The Western blot of endogenous rPLD1 is shown in the lower panel. (B) PLD activity was measured in three clones of Rat2V16, Rat2V25, and Rat2V29 expressing rPLD1-V5 with or without treatment with 10 μg of LPA per ml or 100 nM PMA for 5 or 15 min, respectively. Data are representative of three independent experiments. (C) PLD activity was measured in the Rat2V20 clone, which does not express rPLD1-V5, incubated with or without 10 μg of LPA per ml or 100 nM PMA. Data are representative of two experiments performed in duplicate. Ctrl, control; wt, wild type.

    Journal: Molecular and Cellular Biology

    Article Title: Phospholipase D Activity Is Required for Actin Stress Fiber Formation in Fibroblasts

    doi: 10.1128/MCB.21.12.4055-4066.2001

    Figure Lengend Snippet: The stable expression of V5-tagged rPLD1 in Rat-2 fibroblasts reduces PLD activity. Four G418-resistant clones were selected. (A) The expression of C-terminal V5-tagged rPLD1 was visualized by Western blotting using anti-V5 antibody or an antibody to the C terminus of rPLD1. Lane 1, not Rat-2; lane 2, Rat2V16; lane 3, Rat2V20; lane 4, Rat2V25; lane 5, Rat2V29. The Western blot of endogenous rPLD1 is shown in the lower panel. (B) PLD activity was measured in three clones of Rat2V16, Rat2V25, and Rat2V29 expressing rPLD1-V5 with or without treatment with 10 μg of LPA per ml or 100 nM PMA for 5 or 15 min, respectively. Data are representative of three independent experiments. (C) PLD activity was measured in the Rat2V20 clone, which does not express rPLD1-V5, incubated with or without 10 μg of LPA per ml or 100 nM PMA. Data are representative of two experiments performed in duplicate. Ctrl, control; wt, wild type.

    Article Snippet: Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and the transfer system were obtained from Novex, and the protease inhibitor cocktail and G418 were from Calbiochem.

    Techniques: Expressing, Activity Assay, Clone Assay, Western Blot, Incubation