phosphatase inhibitor cocktail Search Results


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  • 99
    Thermo Fisher halttm phosphatase inhibitor cocktail
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cell Signaling Technology Inc phosphatase inhibitor cocktail
    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Millipore complete phosphatase inhibitor cocktail
    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).
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    Image Search Results


    Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin (  Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies (  Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).

    Journal: eLife

    Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

    doi: 10.7554/eLife.40033

    Figure Lengend Snippet: Cultures of primary human and mouse bone marrow stroma produce microvesicles containing FGF2. Conditioned medium was collected from cultured human bone marrow stroma. Samples were ultracentrifuged and pellets were lysed with 78 μL of Cell Lysis Buffer (Cell Signaling Technologies Inc, Danvers, MA) containing a Complete Mini Protease Inhibitor Cocktail Tablet, Phosphatase Inhibitor Cocktail 2, and Phenylmethanesulfonyl Fluoride (PMSF) solution (Sigma-Aldrich Inc, St Louis, MO) and clarified by centrifugation at 14,000 g, 4°C for 15 min. All samples were loaded on NuPAGE 4–12% Bis-Tris gradient gels, ran in MES buffer (Thermo Fisher Scientific Inc, Waltham, MA), transferred on Immobilon-FL PVDF membranes (Millipore Inc, Billerica, MA), and blocked overnight at 4°C. Following overnight incubation, membranes were incubated with the following primary antibodies: anti-FGFR1, anti-FGF2, anti-CD63, anti-CD9, and anti-actin ( Supplementary file 1 ) overnight at 4°C. The following day membranes were washed and probed with fluorescent IRDye 800CW goat anti-rabbit IgG and IRDye 680RD Goat anti-mouse IgG antibodies ( Supplementary file 1 ). The membranes were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).

    Article Snippet: Immunoblot analysis Treated cells were washed in PBS before adding lysis buffer (Cell Signaling, Danvers, MA, USA) supplemented with Complete protease inhibitor (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail-2 (Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Cell Culture, Lysis, Protease Inhibitor, Centrifugation, Incubation, Imaging