phosphatase inhibitors  (Roche)


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    Structured Review

    Roche phosphatase inhibitors
    Phosphatase Inhibitors, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphatase inhibitors/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phosphatase inhibitors - by Bioz Stars, 2021-06
    86/100 stars

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    Related Articles

    Protease Inhibitor:

    Article Title: Phosphorylation-Mediated Negative Regulation of RIG-I Antiviral Activity ▿
    Article Snippet: .. HEK293T cells were lysed in NP-40 buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% [vol/vol] NP-40, protease inhibitor cocktail [Roche], and Ser/Thr-phosphatase inhibitor cocktail [Sigma]), followed by centrifugation at 13,000 rpm for 20 min. For GST pull-down assays, postcentrifugation supernatants were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction mix was incubated for 3 to 4 h at 4°C. .. For immunoprecipitation, supernatants were incubated with the indicated antibodies at 4°C for 4 to 12 h. Generally, 1 to 2 μg of antibody was added to 1 ml of cell lysate.

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Article Title: How cyclin A destruction escapes the spindle assembly checkpoint
    Article Snippet: The antibody signal was detected using the infrared imaging system (Odyssey; LI-COR) for quantitative immunoblotting. .. APC/C purification and ubiquitylation assays Taxol-treated HeLa cells were resuspended in 20 mM Hepes, pH 7.8, 175 mM NaCl, 2.5 mM MgCl2 , 10% glycerol, 1 mM DTT, 1 mM PMSF, protease inhibitor cocktail (Roche), 2 µM ocadaic acid, 10 nM microcystin LR, and phosphatase inhibitor cocktail II (EMD) and ruptured using nitrogen cavitation. .. APC/C was immunoprecipitated from 10 mg of extract using anti-APC4 antibodies immobilized on protein G (Dynabeads; Invitrogen).

    Centrifugation:

    Article Title: Phosphorylation-Mediated Negative Regulation of RIG-I Antiviral Activity ▿
    Article Snippet: .. HEK293T cells were lysed in NP-40 buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% [vol/vol] NP-40, protease inhibitor cocktail [Roche], and Ser/Thr-phosphatase inhibitor cocktail [Sigma]), followed by centrifugation at 13,000 rpm for 20 min. For GST pull-down assays, postcentrifugation supernatants were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction mix was incubated for 3 to 4 h at 4°C. .. For immunoprecipitation, supernatants were incubated with the indicated antibodies at 4°C for 4 to 12 h. Generally, 1 to 2 μg of antibody was added to 1 ml of cell lysate.

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Binding Assay:

    Article Title: Phosphorylation-Mediated Negative Regulation of RIG-I Antiviral Activity ▿
    Article Snippet: .. HEK293T cells were lysed in NP-40 buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% [vol/vol] NP-40, protease inhibitor cocktail [Roche], and Ser/Thr-phosphatase inhibitor cocktail [Sigma]), followed by centrifugation at 13,000 rpm for 20 min. For GST pull-down assays, postcentrifugation supernatants were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction mix was incubated for 3 to 4 h at 4°C. .. For immunoprecipitation, supernatants were incubated with the indicated antibodies at 4°C for 4 to 12 h. Generally, 1 to 2 μg of antibody was added to 1 ml of cell lysate.

    Incubation:

    Article Title: Phosphorylation-Mediated Negative Regulation of RIG-I Antiviral Activity ▿
    Article Snippet: .. HEK293T cells were lysed in NP-40 buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% [vol/vol] NP-40, protease inhibitor cocktail [Roche], and Ser/Thr-phosphatase inhibitor cocktail [Sigma]), followed by centrifugation at 13,000 rpm for 20 min. For GST pull-down assays, postcentrifugation supernatants were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction mix was incubated for 3 to 4 h at 4°C. .. For immunoprecipitation, supernatants were incubated with the indicated antibodies at 4°C for 4 to 12 h. Generally, 1 to 2 μg of antibody was added to 1 ml of cell lysate.

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Western Blot:

    Article Title: FOXL2, GATA4, and SMAD3 Co-Operatively Modulate Gene Expression, Cell Viability and Apoptosis in Ovarian Granulosa Cell Tumor Cells
    Article Snippet: The over-expression plasmids or the corresponding empty vectors used were; pMT2-GATA4-V5 (V5-tag amino acid sequence GKPIPNPLLGLDST), untagged, V5-tagged, or GFP-tagged pcDNA3.1-FOXL2-WT, pcDNA3.1-FOXL2-C134W, pCDNA3.1-SMAD3 and pCMV-β-Gal. .. Co-immunoprecipitation and Western blotting COS-7 or COV434 cells were lysed 48 h after transfection in 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (pH 7.6) supplemented with protease inhibitors PMSF 1 mM and Complete mini EDTA-free cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitors (Sigma Aldrich, St Louis, MO, USA). .. Clarified lysates were used for Western blotting or immunoprecipitated using Anti-V5 Agarose Affinity Gel (Sigma Aldrich, St Louis, MO, USA) according to instructions.

    Transfection:

    Article Title: FOXL2, GATA4, and SMAD3 Co-Operatively Modulate Gene Expression, Cell Viability and Apoptosis in Ovarian Granulosa Cell Tumor Cells
    Article Snippet: The over-expression plasmids or the corresponding empty vectors used were; pMT2-GATA4-V5 (V5-tag amino acid sequence GKPIPNPLLGLDST), untagged, V5-tagged, or GFP-tagged pcDNA3.1-FOXL2-WT, pcDNA3.1-FOXL2-C134W, pCDNA3.1-SMAD3 and pCMV-β-Gal. .. Co-immunoprecipitation and Western blotting COS-7 or COV434 cells were lysed 48 h after transfection in 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (pH 7.6) supplemented with protease inhibitors PMSF 1 mM and Complete mini EDTA-free cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitors (Sigma Aldrich, St Louis, MO, USA). .. Clarified lysates were used for Western blotting or immunoprecipitated using Anti-V5 Agarose Affinity Gel (Sigma Aldrich, St Louis, MO, USA) according to instructions.

    Article Title: Erk regulation of actin capping and bundling by Eps8 promotes cortex tension and leader bleb-based migration
    Article Snippet: Bands were then resolved on an Odyssey scanner (LI-COR Biosciences). .. Immunoprecipitations 12 hr after transfection of plasmids encoding EGFP or Emerald-tagged mouse Eps8, A375 whole-cell lysates were prepared by scrapeing into ice cold RIPA buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 0.5% deoxycholate and 1% Triton X-100) containing protease and phosphatase inhibitors (Roche). ..

    Activity Assay:

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Lysis:

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Article Title: Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators
    Article Snippet: .. Washed platelets were treated with 5 µg/ml CRP-XL, 5 µM ODN2395, or 5 µM nonmodified ODN2395 for 20 min before addition of ice-cold lysis buffer containing 5 mM EGTA, 1% (vol/vol) Triton X-100, 20 mM Tris-HCl, pH 7.4, and complete protease and phosphatase inhibitor cocktail (Roche). .. Lysates were centrifuged at 1,000 g for 5 min, and supernatants were incubated overnight with either 5 µg/ml mouse anti-Src antibody or mouse anti-Syk antibody (GD11 or 4D10.1; EMD Millipore).

    Purification:

    Article Title: How cyclin A destruction escapes the spindle assembly checkpoint
    Article Snippet: The antibody signal was detected using the infrared imaging system (Odyssey; LI-COR) for quantitative immunoblotting. .. APC/C purification and ubiquitylation assays Taxol-treated HeLa cells were resuspended in 20 mM Hepes, pH 7.8, 175 mM NaCl, 2.5 mM MgCl2 , 10% glycerol, 1 mM DTT, 1 mM PMSF, protease inhibitor cocktail (Roche), 2 µM ocadaic acid, 10 nM microcystin LR, and phosphatase inhibitor cocktail II (EMD) and ruptured using nitrogen cavitation. .. APC/C was immunoprecipitated from 10 mg of extract using anti-APC4 antibodies immobilized on protein G (Dynabeads; Invitrogen).

    Infection:

    Article Title: In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins
    Article Snippet: .. Infected or uninfected subconfluent monolayers of 17Cl-1 cells were lysed with 2× sample buffer containing SDS, protease and phosphatase inhibitors (Roche, Basal, Switzerland), β-mercaptoethanol, and Pierce Universal Nuclease (Thermo Fisher Scientific). .. Boiled cell lysates were separated by SDS-PAGE on polyacrylamide gels.

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    Roche sds lysis buffer
    Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% <t>SDS</t> and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% <t>Bis-Tris)</t> or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p
    Sds Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Roche anti flag conjugated beads sigma aldrich
    MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), <t>immunoprecipitated</t> (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.
    Anti Flag Conjugated Beads Sigma Aldrich, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
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    86
    Roche phosphatase inhibitor phosstop
    Overexpression of KIN10 increased the level of ATG1 phosphorylation. (A) Effect of λ protein phosphatase on the SDS-PAGE mobility of YFP-ATG1a. Proteins were extracted form 7-day-old YFP-ATG1a transgenic plants grown on MS medium. Extracts were treated for 1 h with λ phosphatase (Ppase) with or without the phosphatase inhibitor <t>PhosSTOP</t> and then subjected to immunoblot analysis with anti-GFP antibodies. UN, untreated extracts. (B) Immunoblot analysis of ATG1 phosphorylation in the YFP-ATG1a and YFP-ATG1a/KIN10-OE transgenic plants. Seven-day-old YFP-ATG1a and YFP-ATG1a/KIN10-OE transgenic plants were grown on MS-C medium for the indicated times prior to protein extraction. Crude extracts were subjected to SDS-PAGE and immunoblot analysis with anti-GFP antibodies. The p-YFP-ATG1a and YFP-ATG1a bands are indicated on the right. Coomassie blue-stained total proteins (Rubisco) are shown below the blots to indicate the amount of protein loaded per lane. (C) Quantification of the p-YFP-ATG1a/YFP-ATG1a ratio during carbon starvation by densitometric scans of the immunoblots shown in (B) . The data are means ± SD ( n = 3) calculated from three biological replicates. ∗∗ P
    Phosphatase Inhibitor Phosstop, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% Bis-Tris) or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p

    Journal: PLoS ONE

    Article Title: Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif

    doi: 10.1371/journal.pone.0013672

    Figure Lengend Snippet: Functional characterization of modulation of LRRK2 at T1410. (A) A LanthaScreen assay was used to assess LRRK2 T1410A kinase activity. Titration of wildtype, G2019S, and T1410A (G2019S) LRRK2 proteins demonstrate that mutation T1410A does not affect phosphorylation of LRRKtide. (B) To investigate dimer formation HEK-293T cells were transfected with the indicated LRRK2 constructs. Cell pellets were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication, and 10 ug of protein lysate was loaded onto a native gel (3–12% Bis-Tris) or an SDS gel (7% Tris acetate-SDS), respectively. LRRK2 complexes were visualized with the anti-HA antibody by Western blot with standard ECL. Dimer-sized structures are evident (signal from 480 to 550 kDa), monomeric LRRK2 (278 kDa) is only visible by overexposure (Supplemental Figure S1 , [25] ).Western blots representative of five independent experiments are shown. Normalization of the LRRK2 dimer to total LRRK2 protein (SDS-solubilized) was done using densitometry analysis. ** p

    Article Snippet: Cell pellets were resuspended with either native lysis buffer (phosphate-buffered saline (PBS), pH 7.4, 1x protease and phosphatase inhibitors (Roche Applied Science), lysed by four cycles of freezing and thawing, and analyzed on a 3–12% bis-tris Blue native polyacrylamide gel (Invitrogen) or lysates combined with SDS-lysis buffer (PBS, pH 7.4, 1% SDS, 1xprotease and phosphatase inhibitors (Roche Applied Science) and sonicated with 10% power for 10 s on a Branson dismembranator and analyzed on a 7% Tris acetate SDS gel (Invitrogen).

    Techniques: Functional Assay, Activity Assay, Titration, Mutagenesis, Transfection, Construct, Lysis, Sonication, SDS-Gel, Western Blot

    LRRK2 forms kinase-sensitive high molecular weight conformations. A , cell pellets derived from four lymphoblast cell lines (labeled Lymph1 to -4 ) were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication,

    Journal: The Journal of Biological Chemistry

    Article Title: Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization *

    doi: 10.1074/jbc.M109.025437

    Figure Lengend Snippet: LRRK2 forms kinase-sensitive high molecular weight conformations. A , cell pellets derived from four lymphoblast cell lines (labeled Lymph1 to -4 ) were split equally for lysis by freeze/thaw cycles directly in PBS or lysis with 1% SDS and PBS with sonication,

    Article Snippet: Cell pellets were resuspended with either native lysis buffer (phosphate-buffered saline (PBS), pH 7.4, 1× protease and phosphatase inhibitors (Roche Applied Science)), lysed by four cycles of freezing and thawing, and analyzed on a 3–12% bis-tris Blue native polyacrylamide gel (Invitrogen) or lysates combined with SDS-lysis buffer (PBS, pH 7.4, 1% SDS, 1× protease and phosphatase inhibitors (Roche Applied Science)) and sonicated with 10% power for 10 s on a Branson dismembranator and analyzed on a 7% Tris acetate SDS gel (Invitrogen).

    Techniques: Molecular Weight, Derivative Assay, Labeling, Lysis, Sonication

    MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), immunoprecipitated (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.

    Journal: EBioMedicine

    Article Title: Mitochondrial fission factor is a novel Myc-dependent regulator of mitochondrial permeability in cancer

    doi: 10.1016/j.ebiom.2019.09.017

    Figure Lengend Snippet: MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), immunoprecipitated (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.

    Article Snippet: 2.6 Immunoprecipitation (IP) Cell extracts prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA containing 1% CHAPS, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) and Phosphatase Inhibitor Cocktail PhosSTOP (Roche) were immunoprecipitated with anti-Flag-conjugated beads (Sigma-Aldrich) and processed as described [ ].

    Techniques: Permeability, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Flow Cytometry, Cytometry, Fluorescence, Incubation, Staining

    Overexpression of KIN10 increased the level of ATG1 phosphorylation. (A) Effect of λ protein phosphatase on the SDS-PAGE mobility of YFP-ATG1a. Proteins were extracted form 7-day-old YFP-ATG1a transgenic plants grown on MS medium. Extracts were treated for 1 h with λ phosphatase (Ppase) with or without the phosphatase inhibitor PhosSTOP and then subjected to immunoblot analysis with anti-GFP antibodies. UN, untreated extracts. (B) Immunoblot analysis of ATG1 phosphorylation in the YFP-ATG1a and YFP-ATG1a/KIN10-OE transgenic plants. Seven-day-old YFP-ATG1a and YFP-ATG1a/KIN10-OE transgenic plants were grown on MS-C medium for the indicated times prior to protein extraction. Crude extracts were subjected to SDS-PAGE and immunoblot analysis with anti-GFP antibodies. The p-YFP-ATG1a and YFP-ATG1a bands are indicated on the right. Coomassie blue-stained total proteins (Rubisco) are shown below the blots to indicate the amount of protein loaded per lane. (C) Quantification of the p-YFP-ATG1a/YFP-ATG1a ratio during carbon starvation by densitometric scans of the immunoblots shown in (B) . The data are means ± SD ( n = 3) calculated from three biological replicates. ∗∗ P

    Journal: Frontiers in Plant Science

    Article Title: The AMP-Activated Protein Kinase KIN10 Is Involved in the Regulation of Autophagy in Arabidopsis

    doi: 10.3389/fpls.2017.01201

    Figure Lengend Snippet: Overexpression of KIN10 increased the level of ATG1 phosphorylation. (A) Effect of λ protein phosphatase on the SDS-PAGE mobility of YFP-ATG1a. Proteins were extracted form 7-day-old YFP-ATG1a transgenic plants grown on MS medium. Extracts were treated for 1 h with λ phosphatase (Ppase) with or without the phosphatase inhibitor PhosSTOP and then subjected to immunoblot analysis with anti-GFP antibodies. UN, untreated extracts. (B) Immunoblot analysis of ATG1 phosphorylation in the YFP-ATG1a and YFP-ATG1a/KIN10-OE transgenic plants. Seven-day-old YFP-ATG1a and YFP-ATG1a/KIN10-OE transgenic plants were grown on MS-C medium for the indicated times prior to protein extraction. Crude extracts were subjected to SDS-PAGE and immunoblot analysis with anti-GFP antibodies. The p-YFP-ATG1a and YFP-ATG1a bands are indicated on the right. Coomassie blue-stained total proteins (Rubisco) are shown below the blots to indicate the amount of protein loaded per lane. (C) Quantification of the p-YFP-ATG1a/YFP-ATG1a ratio during carbon starvation by densitometric scans of the immunoblots shown in (B) . The data are means ± SD ( n = 3) calculated from three biological replicates. ∗∗ P

    Article Snippet: The supernatant was incubated with λ protein phosphatase (New England Biolabs) with or without addition of phosphatase inhibitor PhosSTOP (Roche) for 30 min at 30°C.

    Techniques: Over Expression, SDS Page, Transgenic Assay, Mass Spectrometry, Protein Extraction, Staining, Western Blot