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Roche ethylene glycol tetraacetic acid egta
Ethylene Glycol Tetraacetic Acid Egta, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 15 article reviews
Price from $9.99 to $1999.99
ethylene glycol tetraacetic acid egta - by Bioz Stars, 2020-07
92/100 stars

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Related Articles

Transfection:

Article Title: Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment
Article Snippet: .. Transfected N2a cells were rinsed twice with ice-cold PBS, lysed in homogenization buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% nonyl phenoxy polyethoxylethanol (NP-40), 10% glycerol, 1 mM ethylene glycol tetraacetic acid (EGTA), 20 mM NaF, 1 mM Na3 VO4 , 5 μM OA and protease inhibitor cocktail (Roche Applied Science,Basel, Switzerland) and incubated on ice for 30 min. After centrifugation at 16000×g at 4 °C for 20 min, the supernatant was collected and precleared with Dynabeads Protein G (Thermo Fisher Scientific; Dreieich, Germany) for 1 h at 4 °C. .. The lysates were incubated with control IgG or anti-His or DA9 antibodies overnight with constant rotation at 4 °C.

Radio Immunoprecipitation:

Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
Article Snippet: .. Western blot analysis Whole-protein extracts from cells or tumors were prepared after cell scrapping or tissue homogenization, respectively, in radioimmunoprecipitation assay buffer [RIPA buffer: 150 mM sodium chloride (NaCl), 50 mM Tris hydrochloride (Tris-HCl), pH 7.5, 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 0.5% sodium deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), 2 mM phenylmethanesulfonyl (PMSF), 2 mM iodoacetamide (IAD), and 1 × protease inhibitor cocktail (Roche)]. .. After centrifugation at 16,000×g for 10 min at 4 °C, supernatants were used for protein quantification as determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), followed by denaturation of the sample with Laemmli buffer.

Homogenization:

Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
Article Snippet: .. Western blot analysis Whole-protein extracts from cells or tumors were prepared after cell scrapping or tissue homogenization, respectively, in radioimmunoprecipitation assay buffer [RIPA buffer: 150 mM sodium chloride (NaCl), 50 mM Tris hydrochloride (Tris-HCl), pH 7.5, 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 0.5% sodium deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), 2 mM phenylmethanesulfonyl (PMSF), 2 mM iodoacetamide (IAD), and 1 × protease inhibitor cocktail (Roche)]. .. After centrifugation at 16,000×g for 10 min at 4 °C, supernatants were used for protein quantification as determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), followed by denaturation of the sample with Laemmli buffer.

Article Title: Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment
Article Snippet: .. Transfected N2a cells were rinsed twice with ice-cold PBS, lysed in homogenization buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% nonyl phenoxy polyethoxylethanol (NP-40), 10% glycerol, 1 mM ethylene glycol tetraacetic acid (EGTA), 20 mM NaF, 1 mM Na3 VO4 , 5 μM OA and protease inhibitor cocktail (Roche Applied Science,Basel, Switzerland) and incubated on ice for 30 min. After centrifugation at 16000×g at 4 °C for 20 min, the supernatant was collected and precleared with Dynabeads Protein G (Thermo Fisher Scientific; Dreieich, Germany) for 1 h at 4 °C. .. The lysates were incubated with control IgG or anti-His or DA9 antibodies overnight with constant rotation at 4 °C.

Protease Inhibitor:

Article Title: Photoexcited Toluidine Blue Inhibits Tau Aggregation in Alzheimer’s Disease
Article Snippet: .. Other chemicals such as ampicillin, NaCl, KCl, Na2 HPO4 , KH2 PO4 , ethylene glycol tetraacetic acid (EGTA), MgCl2 , phenylmethylsulfonyl fluoride (PMSF), ammonium acetate, and bovine serum albumin (BSA) were obtained from MP and protease inhibitor cocktail was obtained from Roche. .. Copper-coated carbon grids were purchased from Ted Pella, Inc. Advanced DMEM/F-12 media, fetal bovine serum (FBS), pensterp cocktail, and anti-anti were purchased from Gibco.

Article Title: Nutrient ingestion increased mTOR signaling, but not hVps34 activity in human skeletal muscle after sprint exercise
Article Snippet: .. Western blot Muscle samples (∼30 mg) were homogenized on ice using glass homogenizers in ice-cold buffer (20 μL mg−1 wet weight) containing 20 mmol L−1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 1 mmol L−1 ethylene diamine tetraacetic acid (EDTA) (pH 7.4), 1 mmol L−1 Na3 VO4 , 5 mmol L−1 ethylene glycol tetraacetic acid (EGTA) (pH 7.4), 10 mmol L−1 MgCl2 , 50 mmol L−1 β-glycerophosphate, 2 mmol L−1 dithiothreitol, 1% Triton X-100, and one tablet (per 10 mL) of Complete mini protease inhibitor tablets (Roche, Diagnostics, Indianapolis, IN) diluted in Milli Q® water (Millipore, Solna, Sweden). .. Homogenates were rotated (RM5 assistant 348, rotating mixer, Karl Hecht, Sondheim, Germany) for 60 min at 4°C and centrifuged at 15,000g for 10 min at 4°C to remove cell debris.

Article Title: A role for the Smc3 hinge domain in the maintenance of sister chromatid cohesion
Article Snippet: .. Cells were pelleted and resuspended in lysis buffer consisting of 25 mM HEPES, pH 8.0, 2 mM MgCl2 , 100 μM EDTA, 500 μM egtazic acid (EGTA), 1% NP-40, 150 mM KCl, 15% glycerol, Complete-Mini EDTA-free protease inhibitor cocktail (Roche), 10 mM sodium butyrate, and 20 mM beta-glycerophosphate. .. Cells were incubated in buffer for 30 min on ice, and then glass beads were added to a 1:1 volume ratio before bead beating for 3 min. Lysates were pelleted at 14,000 rpm for 10 min at 4°C, and protein concentration was measured using Coomassie Brilliant Blue.

Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
Article Snippet: .. Western blot analysis Whole-protein extracts from cells or tumors were prepared after cell scrapping or tissue homogenization, respectively, in radioimmunoprecipitation assay buffer [RIPA buffer: 150 mM sodium chloride (NaCl), 50 mM Tris hydrochloride (Tris-HCl), pH 7.5, 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 0.5% sodium deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), 2 mM phenylmethanesulfonyl (PMSF), 2 mM iodoacetamide (IAD), and 1 × protease inhibitor cocktail (Roche)]. .. After centrifugation at 16,000×g for 10 min at 4 °C, supernatants were used for protein quantification as determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), followed by denaturation of the sample with Laemmli buffer.

Article Title: Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment
Article Snippet: .. Transfected N2a cells were rinsed twice with ice-cold PBS, lysed in homogenization buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% nonyl phenoxy polyethoxylethanol (NP-40), 10% glycerol, 1 mM ethylene glycol tetraacetic acid (EGTA), 20 mM NaF, 1 mM Na3 VO4 , 5 μM OA and protease inhibitor cocktail (Roche Applied Science,Basel, Switzerland) and incubated on ice for 30 min. After centrifugation at 16000×g at 4 °C for 20 min, the supernatant was collected and precleared with Dynabeads Protein G (Thermo Fisher Scientific; Dreieich, Germany) for 1 h at 4 °C. .. The lysates were incubated with control IgG or anti-His or DA9 antibodies overnight with constant rotation at 4 °C.

Centrifugation:

Article Title: Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment
Article Snippet: .. Transfected N2a cells were rinsed twice with ice-cold PBS, lysed in homogenization buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% nonyl phenoxy polyethoxylethanol (NP-40), 10% glycerol, 1 mM ethylene glycol tetraacetic acid (EGTA), 20 mM NaF, 1 mM Na3 VO4 , 5 μM OA and protease inhibitor cocktail (Roche Applied Science,Basel, Switzerland) and incubated on ice for 30 min. After centrifugation at 16000×g at 4 °C for 20 min, the supernatant was collected and precleared with Dynabeads Protein G (Thermo Fisher Scientific; Dreieich, Germany) for 1 h at 4 °C. .. The lysates were incubated with control IgG or anti-His or DA9 antibodies overnight with constant rotation at 4 °C.

Incubation:

Article Title: Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes
Article Snippet: .. We incubated cells on ice for 45 min in a lysis buffer containing 50 mM trizma hydrochloride (Tris–HCl), pH 7.5, 50 mM mannitol, 2 mM ethylene glycol tetraacetic acid (EGTA), 2 mM dithiothreitol (DTT), 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), and 1% (w/v) aprotinin (Roche Diagnostics, Indianapolis, IN) and subsequently disrupted the cells using a Dounce homogenizer (30 strokes with pestle A). .. We removed undisrupted cells and nuclear debris by centrifugation at 500 × g for 10 min. We diluted the supernatant 2-fold in resuspension buffer containing 50 mM Tris–HCl (pH 7.5), 300 mM mannitol, 1 mM EGTA, 1 mM DTT, 1 mM AEBSF, and 1% (w/v) aprotinin.

Article Title: Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment
Article Snippet: .. Transfected N2a cells were rinsed twice with ice-cold PBS, lysed in homogenization buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% nonyl phenoxy polyethoxylethanol (NP-40), 10% glycerol, 1 mM ethylene glycol tetraacetic acid (EGTA), 20 mM NaF, 1 mM Na3 VO4 , 5 μM OA and protease inhibitor cocktail (Roche Applied Science,Basel, Switzerland) and incubated on ice for 30 min. After centrifugation at 16000×g at 4 °C for 20 min, the supernatant was collected and precleared with Dynabeads Protein G (Thermo Fisher Scientific; Dreieich, Germany) for 1 h at 4 °C. .. The lysates were incubated with control IgG or anti-His or DA9 antibodies overnight with constant rotation at 4 °C.

Activity Assay:

Article Title: Chronic Microdose Lithium Treatment Prevented Memory Loss and Neurohistopathological Changes in a Transgenic Mouse Model of Alzheimer's Disease
Article Snippet: .. Evaluation of synaptophysin density and GSK-3β activity Cortical and hippocampal tissues were homogenized in lysis buffer containing 50 mM Tris–HCl (pH 7.4), 0.1% Triton X-100, 4 mM ethylene glycol tetraacetic acid (EGTA), 10 mM ethylenediamine tetracetic acid (EDTA), a tablet with protease inhibitors (Complete EDTA-free, Roche Diagnostics, Germany) and a phosphatase inhibitor cocktail tablet (PhosStop, Roche Diagnostics, Germany). .. Proteins (7 or 50 μg/lane, according to the protein of interest) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes.

Lysis:

Article Title: A role for the Smc3 hinge domain in the maintenance of sister chromatid cohesion
Article Snippet: .. Cells were pelleted and resuspended in lysis buffer consisting of 25 mM HEPES, pH 8.0, 2 mM MgCl2 , 100 μM EDTA, 500 μM egtazic acid (EGTA), 1% NP-40, 150 mM KCl, 15% glycerol, Complete-Mini EDTA-free protease inhibitor cocktail (Roche), 10 mM sodium butyrate, and 20 mM beta-glycerophosphate. .. Cells were incubated in buffer for 30 min on ice, and then glass beads were added to a 1:1 volume ratio before bead beating for 3 min. Lysates were pelleted at 14,000 rpm for 10 min at 4°C, and protein concentration was measured using Coomassie Brilliant Blue.

Article Title: Chronic Microdose Lithium Treatment Prevented Memory Loss and Neurohistopathological Changes in a Transgenic Mouse Model of Alzheimer's Disease
Article Snippet: .. Evaluation of synaptophysin density and GSK-3β activity Cortical and hippocampal tissues were homogenized in lysis buffer containing 50 mM Tris–HCl (pH 7.4), 0.1% Triton X-100, 4 mM ethylene glycol tetraacetic acid (EGTA), 10 mM ethylenediamine tetracetic acid (EDTA), a tablet with protease inhibitors (Complete EDTA-free, Roche Diagnostics, Germany) and a phosphatase inhibitor cocktail tablet (PhosStop, Roche Diagnostics, Germany). .. Proteins (7 or 50 μg/lane, according to the protein of interest) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes.

Article Title: Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes
Article Snippet: .. We incubated cells on ice for 45 min in a lysis buffer containing 50 mM trizma hydrochloride (Tris–HCl), pH 7.5, 50 mM mannitol, 2 mM ethylene glycol tetraacetic acid (EGTA), 2 mM dithiothreitol (DTT), 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), and 1% (w/v) aprotinin (Roche Diagnostics, Indianapolis, IN) and subsequently disrupted the cells using a Dounce homogenizer (30 strokes with pestle A). .. We removed undisrupted cells and nuclear debris by centrifugation at 500 × g for 10 min. We diluted the supernatant 2-fold in resuspension buffer containing 50 mM Tris–HCl (pH 7.5), 300 mM mannitol, 1 mM EGTA, 1 mM DTT, 1 mM AEBSF, and 1% (w/v) aprotinin.

Western Blot:

Article Title: Nutrient ingestion increased mTOR signaling, but not hVps34 activity in human skeletal muscle after sprint exercise
Article Snippet: .. Western blot Muscle samples (∼30 mg) were homogenized on ice using glass homogenizers in ice-cold buffer (20 μL mg−1 wet weight) containing 20 mmol L−1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 1 mmol L−1 ethylene diamine tetraacetic acid (EDTA) (pH 7.4), 1 mmol L−1 Na3 VO4 , 5 mmol L−1 ethylene glycol tetraacetic acid (EGTA) (pH 7.4), 10 mmol L−1 MgCl2 , 50 mmol L−1 β-glycerophosphate, 2 mmol L−1 dithiothreitol, 1% Triton X-100, and one tablet (per 10 mL) of Complete mini protease inhibitor tablets (Roche, Diagnostics, Indianapolis, IN) diluted in Milli Q® water (Millipore, Solna, Sweden). .. Homogenates were rotated (RM5 assistant 348, rotating mixer, Karl Hecht, Sondheim, Germany) for 60 min at 4°C and centrifuged at 15,000g for 10 min at 4°C to remove cell debris.

Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
Article Snippet: .. Western blot analysis Whole-protein extracts from cells or tumors were prepared after cell scrapping or tissue homogenization, respectively, in radioimmunoprecipitation assay buffer [RIPA buffer: 150 mM sodium chloride (NaCl), 50 mM Tris hydrochloride (Tris-HCl), pH 7.5, 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 0.5% sodium deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), 2 mM phenylmethanesulfonyl (PMSF), 2 mM iodoacetamide (IAD), and 1 × protease inhibitor cocktail (Roche)]. .. After centrifugation at 16,000×g for 10 min at 4 °C, supernatants were used for protein quantification as determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), followed by denaturation of the sample with Laemmli buffer.

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  • 90
    Roche p70s6k1 panpkb
    Saturation time course of activity assays carried out from pooled human skeletal muscle protein lysate. R 2 values are as follows: AMPK = 0.969; <t>panPKB</t> = 0.982; and <t>p70S6K1</t> = 0.856. All data are expressed as means ± SD.
    P70s6k1 Panpkb, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p70s6k1 panpkb/product/Roche
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p70s6k1 panpkb - by Bioz Stars, 2020-07
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    93
    Roche np 40 buffer
    Feedback activation of PI3Kα following PI3Kδ inhibition depends on increased BCR signaling A. TMD8 was exposed over different time periods to CAL-101. Western blot indicates increased proximal BCR signaling following PI3Kδ inhibition. B. TMD8 and Ly10 were treated with 200nM CAL-101, 50nM Dasatinib (src inhibitor), 1000nM PRT062607 (Syk inhibitor) at the indicated time points and harvested at 2hr and 24hr. Results indicates that rebound PI3K reactivation following PI3Kδ inhibition is sensitive to Src and Syk inhibition. C. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with <t>NP-40</t> lysis buffer, immunoprecipitated with BCAP and probed for the indicated proteins. D. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with CD19 and probed for the indicated proteins.
    Np 40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np 40 buffer/product/Roche
    Average 93 stars, based on 9 article reviews
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    egta  (Roche)
    94
    Roche egta
    Interactions among S. pombe GST-Myo1p-23A, GST-Wsp1p-VCA, Arp2/3 complex, Vrp1p-His, and actin monomers. Soluble protein ligands were incubated with a range of concentrations of GST-Myo1p-23A or GST-Wsp1p-VCA immobilized on glutathione-Sepharose or Vrp1p-His bound to Ni-NTA resin. Beads and bound ligand were pelleted and concentrations of bound ligands calculated from measurements of unbound ligands in the supernatants by SDS-PAGE, staining with Coomassie blue and densitometry. (A and B) Binding of 0.3 μM Arp2/3 complex to (A) GST-Myo1p-23A or (B) GST-Wsp1p-VCA immobilized on glutathione beads. (C) Binding of 0.5 μM Vrp1p-His to immobilized GST-Myo1p-23A. Conditions (A–C): 10 mM imidazole, pH 7.0, 50 mM <t>KCl,</t> 1 mM MgCl 2 , 1 mM <t>EGTA,</t> 0.1 mM ATP, and 1 mM DTT. (D) Binding of 0.5 μM actin monomers to Vrp1p-His immobilized on beads. Conditions (D): 2 mM Tris-HCl, pH 8.0, 0.2 mM ATP, and 0.1 mM CaCl 2 . Equilibrium dissociation constants were determined by fitting data to binding isotherms (solid lines).
    Egta, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 256 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche radioimmune precipitation buffer
    ASK1 expression is reduced in the presence of overexpressed CHIP. COS-7 cells were transiently transfected with HA-ASK1 with or without CHIP (wild type and H260Q). Twenty-four hours after transfection, cells were lysed in <t>radioimmune</t> precipitation
    Radioimmune Precipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 29 article reviews
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    Image Search Results


    Saturation time course of activity assays carried out from pooled human skeletal muscle protein lysate. R 2 values are as follows: AMPK = 0.969; panPKB = 0.982; and p70S6K1 = 0.856. All data are expressed as means ± SD.

    Journal: Journal of Applied Physiology

    Article Title: Application of the [γ-32P] ATP kinase assay to study anabolic signaling in human skeletal muscle

    doi: 10.1152/japplphysiol.01072.2013

    Figure Lengend Snippet: Saturation time course of activity assays carried out from pooled human skeletal muscle protein lysate. R 2 values are as follows: AMPK = 0.969; panPKB = 0.982; and p70S6K1 = 0.856. All data are expressed as means ± SD.

    Article Snippet: All KA were carried out by IP either for 2 h at 4°C or overnight at 4°C in homogenization buffer {AMPK [50 mM Tris·HCl pH 7.25, 150 mM NaCl, 50 mM NaF, 5 mM NaPPi, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 5 μg/ml soybean trypsin inhibitor, 1% (vol/vol) Triton X-100] and p70S6K1/panPKB [50 mM Tris·HCl pH 7.5, 0.1 mM EGTA, 1 mM EDTA, 1% (vol/vol) Triton X-100, 50 mM NaF, 5 mM NaPPi, 0.27 M sucrose, 0.1% β-mercaptoethanol, 1 mM Na3 (OV)4 , and 1 Complete (Roche) protease inhibitor tablet per 10 ml]}.

    Techniques: Activity Assay

    Serial immunoprecipitation (IP) validation. IPs were set up to immunoprecipitate panPKB alone or p70S6K1 immunoprecipitated prior to immunoprecipitating with panPKB. *Significantly different from both control (Con) conditions. All data are expressed as

    Journal: Journal of Applied Physiology

    Article Title: Application of the [γ-32P] ATP kinase assay to study anabolic signaling in human skeletal muscle

    doi: 10.1152/japplphysiol.01072.2013

    Figure Lengend Snippet: Serial immunoprecipitation (IP) validation. IPs were set up to immunoprecipitate panPKB alone or p70S6K1 immunoprecipitated prior to immunoprecipitating with panPKB. *Significantly different from both control (Con) conditions. All data are expressed as

    Article Snippet: All KA were carried out by IP either for 2 h at 4°C or overnight at 4°C in homogenization buffer {AMPK [50 mM Tris·HCl pH 7.25, 150 mM NaCl, 50 mM NaF, 5 mM NaPPi, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 5 μg/ml soybean trypsin inhibitor, 1% (vol/vol) Triton X-100] and p70S6K1/panPKB [50 mM Tris·HCl pH 7.5, 0.1 mM EGTA, 1 mM EDTA, 1% (vol/vol) Triton X-100, 50 mM NaF, 5 mM NaPPi, 0.27 M sucrose, 0.1% β-mercaptoethanol, 1 mM Na3 (OV)4 , and 1 Complete (Roche) protease inhibitor tablet per 10 ml]}.

    Techniques: Immunoprecipitation

    Feedback activation of PI3Kα following PI3Kδ inhibition depends on increased BCR signaling A. TMD8 was exposed over different time periods to CAL-101. Western blot indicates increased proximal BCR signaling following PI3Kδ inhibition. B. TMD8 and Ly10 were treated with 200nM CAL-101, 50nM Dasatinib (src inhibitor), 1000nM PRT062607 (Syk inhibitor) at the indicated time points and harvested at 2hr and 24hr. Results indicates that rebound PI3K reactivation following PI3Kδ inhibition is sensitive to Src and Syk inhibition. C. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with BCAP and probed for the indicated proteins. D. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with CD19 and probed for the indicated proteins.

    Journal: Oncotarget

    Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma

    doi: 10.18632/oncotarget.20864

    Figure Lengend Snippet: Feedback activation of PI3Kα following PI3Kδ inhibition depends on increased BCR signaling A. TMD8 was exposed over different time periods to CAL-101. Western blot indicates increased proximal BCR signaling following PI3Kδ inhibition. B. TMD8 and Ly10 were treated with 200nM CAL-101, 50nM Dasatinib (src inhibitor), 1000nM PRT062607 (Syk inhibitor) at the indicated time points and harvested at 2hr and 24hr. Results indicates that rebound PI3K reactivation following PI3Kδ inhibition is sensitive to Src and Syk inhibition. C. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with BCAP and probed for the indicated proteins. D. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with CD19 and probed for the indicated proteins.

    Article Snippet: Immunoblot Cells were pelleted by centrifugation, washed with ice-cold PBS and lysed on ice for 20 min in modified NP-40 buffer (10mM/1mM KPO4/EDTA pH 7.05, 5mM EGTA pH 7.2, 10mM MgCl2, 50mM glycerophosphate pH 7.2, 0.5% NP-40, 0.1% Brij-35) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 1mM DTT, 1mM Va3VO4 and 1mM PMSF.

    Techniques: Activation Assay, Inhibition, Western Blot, Lysis, Immunoprecipitation

    Interactions among S. pombe GST-Myo1p-23A, GST-Wsp1p-VCA, Arp2/3 complex, Vrp1p-His, and actin monomers. Soluble protein ligands were incubated with a range of concentrations of GST-Myo1p-23A or GST-Wsp1p-VCA immobilized on glutathione-Sepharose or Vrp1p-His bound to Ni-NTA resin. Beads and bound ligand were pelleted and concentrations of bound ligands calculated from measurements of unbound ligands in the supernatants by SDS-PAGE, staining with Coomassie blue and densitometry. (A and B) Binding of 0.3 μM Arp2/3 complex to (A) GST-Myo1p-23A or (B) GST-Wsp1p-VCA immobilized on glutathione beads. (C) Binding of 0.5 μM Vrp1p-His to immobilized GST-Myo1p-23A. Conditions (A–C): 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM ATP, and 1 mM DTT. (D) Binding of 0.5 μM actin monomers to Vrp1p-His immobilized on beads. Conditions (D): 2 mM Tris-HCl, pH 8.0, 0.2 mM ATP, and 0.1 mM CaCl 2 . Equilibrium dissociation constants were determined by fitting data to binding isotherms (solid lines).

    Journal: The Journal of Cell Biology

    Article Title: Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast

    doi: 10.1083/jcb.200502053

    Figure Lengend Snippet: Interactions among S. pombe GST-Myo1p-23A, GST-Wsp1p-VCA, Arp2/3 complex, Vrp1p-His, and actin monomers. Soluble protein ligands were incubated with a range of concentrations of GST-Myo1p-23A or GST-Wsp1p-VCA immobilized on glutathione-Sepharose or Vrp1p-His bound to Ni-NTA resin. Beads and bound ligand were pelleted and concentrations of bound ligands calculated from measurements of unbound ligands in the supernatants by SDS-PAGE, staining with Coomassie blue and densitometry. (A and B) Binding of 0.3 μM Arp2/3 complex to (A) GST-Myo1p-23A or (B) GST-Wsp1p-VCA immobilized on glutathione beads. (C) Binding of 0.5 μM Vrp1p-His to immobilized GST-Myo1p-23A. Conditions (A–C): 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM ATP, and 1 mM DTT. (D) Binding of 0.5 μM actin monomers to Vrp1p-His immobilized on beads. Conditions (D): 2 mM Tris-HCl, pH 8.0, 0.2 mM ATP, and 0.1 mM CaCl 2 . Equilibrium dissociation constants were determined by fitting data to binding isotherms (solid lines).

    Article Snippet: Protein purification Native S. pombe Arp2/3 complex was purified from protease-deficient TM011 cells resuspended in buffer U (50 mM Hepes, pH 7.5, 100 mM KCl, 3 mM MgCl2 , 1 mM EGTA, 0.1 mM ATP, and 1 mM DTT) containing Complete (Roche) protease inhibitors and ruptured using a Microfluidizer (model M-110S; Microfluidics).

    Techniques: Incubation, SDS Page, Staining, Binding Assay

    Actin polymerization assays for the activation of S. pombe Arp2/3 complex by WASP-family VCA segments, S. pombe Myo1p tail, and verprolin. Polymerization of 4 μM Mg-ATP actin (5% pyrene-labeled) alone or with 10 nM Arp2/3 complex and activator proteins was measured by the fluorescence of pyrene-labeled actin. Conditions: 10 mM imidazole, pH 7.0, 50 mM KCl, (A–C) 5 mM or (D) 21 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN 3 , and 20 μM CaCl 2 . (A) Time course of polymerization of actin (•) alone or with (○) GST-Myo1p-23A + verprolin, (♦) Arp2/3 complex, (⋄) Arp2/3 complex + verprolin, (▪) Arp2/3 complex + GST-Myo1p-23A, (□) Arp2/3 complex + GST-Myo1p-23A + verprolin, (▴) Arp2/3 complex + GST-Wsp1p-VCA, (▵) Arp2/3 complex + GST-Wsp1p-VCA + verprolin at the following concentrations: 200 nM GST-Myo1p-23A, 50 nM GST-Wsp1p-VCA, 100 nM verprolin. Inset: (○) Concentration of actin filament ends generated by 10 nM Arp2/3 complex and 50 nM GST-Myo1p-23A as a function of the concentration of verprolin; (▪) reaction without GST-Myo1p-23A. (B) Dependence of the concentration of actin filament ends generated by 10 nM Arp2/3 complex on the concentrations of (▪) GST-Myo1p-23A, (□) GST-Myo1p-23A with 500 nM Vrp1p, (▴) GST-Wsp1p-VCA and (○) bovine GST-N-WASP-VVCA. Concentrations of ends were calculated from the polymerization rate when half of the actin was polymerized. (C) Effect of preexisting actin filaments on the time course of actin polymerization stimulated by 10 nM Arp2/3 complex and 200 nM GST-Myo1p-23A alone or with 200 nM verprolin. Buffer or 0.2 μM F-actin were added at the onset of polymerization reactions: actin with (•) buffer or (○) F-actin added to actin alone, (▪) buffer or (□) F-actin added to Arp2/3 complex + GST-Myo1p-23A, (▴) buffer (▵) or F-actin added to Arp2/3 complex + GST-Myo1p-23A + verprolin. (D) Myo1p tail constructs lacking either the TH2 or A domain inhibit actin polymerization stimulated by 10 nM Arp2/3 complex and 200 nM GST-Myo1p-23A in both the absence and presence of 700 nM verprolin. 1 μM GST-Myo1p-3A or GST-Myo1p-23 was added before reaction onset. Reactions contained (•) actin alone or actin with (▪) buffer, (▴) GST-Myo1p-3A, or (▾) GST-Myo1p-23 added to Arp2/3 complex + GST-Myo1p-23A; or actin with (□) buffer, (▵) GST-Myo1p-3A, (▿) GST-Myo1p-23 added to Arp2/3 complex + GST-Myo1p-23A + verprolin before reaction onset.

    Journal: The Journal of Cell Biology

    Article Title: Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast

    doi: 10.1083/jcb.200502053

    Figure Lengend Snippet: Actin polymerization assays for the activation of S. pombe Arp2/3 complex by WASP-family VCA segments, S. pombe Myo1p tail, and verprolin. Polymerization of 4 μM Mg-ATP actin (5% pyrene-labeled) alone or with 10 nM Arp2/3 complex and activator proteins was measured by the fluorescence of pyrene-labeled actin. Conditions: 10 mM imidazole, pH 7.0, 50 mM KCl, (A–C) 5 mM or (D) 21 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN 3 , and 20 μM CaCl 2 . (A) Time course of polymerization of actin (•) alone or with (○) GST-Myo1p-23A + verprolin, (♦) Arp2/3 complex, (⋄) Arp2/3 complex + verprolin, (▪) Arp2/3 complex + GST-Myo1p-23A, (□) Arp2/3 complex + GST-Myo1p-23A + verprolin, (▴) Arp2/3 complex + GST-Wsp1p-VCA, (▵) Arp2/3 complex + GST-Wsp1p-VCA + verprolin at the following concentrations: 200 nM GST-Myo1p-23A, 50 nM GST-Wsp1p-VCA, 100 nM verprolin. Inset: (○) Concentration of actin filament ends generated by 10 nM Arp2/3 complex and 50 nM GST-Myo1p-23A as a function of the concentration of verprolin; (▪) reaction without GST-Myo1p-23A. (B) Dependence of the concentration of actin filament ends generated by 10 nM Arp2/3 complex on the concentrations of (▪) GST-Myo1p-23A, (□) GST-Myo1p-23A with 500 nM Vrp1p, (▴) GST-Wsp1p-VCA and (○) bovine GST-N-WASP-VVCA. Concentrations of ends were calculated from the polymerization rate when half of the actin was polymerized. (C) Effect of preexisting actin filaments on the time course of actin polymerization stimulated by 10 nM Arp2/3 complex and 200 nM GST-Myo1p-23A alone or with 200 nM verprolin. Buffer or 0.2 μM F-actin were added at the onset of polymerization reactions: actin with (•) buffer or (○) F-actin added to actin alone, (▪) buffer or (□) F-actin added to Arp2/3 complex + GST-Myo1p-23A, (▴) buffer (▵) or F-actin added to Arp2/3 complex + GST-Myo1p-23A + verprolin. (D) Myo1p tail constructs lacking either the TH2 or A domain inhibit actin polymerization stimulated by 10 nM Arp2/3 complex and 200 nM GST-Myo1p-23A in both the absence and presence of 700 nM verprolin. 1 μM GST-Myo1p-3A or GST-Myo1p-23 was added before reaction onset. Reactions contained (•) actin alone or actin with (▪) buffer, (▴) GST-Myo1p-3A, or (▾) GST-Myo1p-23 added to Arp2/3 complex + GST-Myo1p-23A; or actin with (□) buffer, (▵) GST-Myo1p-3A, (▿) GST-Myo1p-23 added to Arp2/3 complex + GST-Myo1p-23A + verprolin before reaction onset.

    Article Snippet: Protein purification Native S. pombe Arp2/3 complex was purified from protease-deficient TM011 cells resuspended in buffer U (50 mM Hepes, pH 7.5, 100 mM KCl, 3 mM MgCl2 , 1 mM EGTA, 0.1 mM ATP, and 1 mM DTT) containing Complete (Roche) protease inhibitors and ruptured using a Microfluidizer (model M-110S; Microfluidics).

    Techniques: Activation Assay, Labeling, Fluorescence, Concentration Assay, Generated, Construct

    Fluorescence microscopy of the products of actin polymerization reactions. Conditions: 4 μM monomeric actin (5% pyrene), 10 mM imidazole, pH 7.0, 50 mM KCl, 5 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN 3 , and 20 μM CaCl 2 . After mixing the reactants, the samples were split into two: (A) pyrene fluorescence was used to monitor the polymerization of one aliquot; (B–F) 4 μM rhodamine-phalloidin was added to the other aliquot. When each reaction was 80% complete, the aliquot with rhodamine-phalloidin was diluted 400 times into microscopy buffer (10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl 2 , 100 mM DTT, 20 μg/ml catalase, 100 μg/ml glucose oxidase, 3 mg/ml glucose, and 0.5% methylcellulose) and 2 μl were applied to the nitrocellulose-coated 22 × 22-mm coverslip. (A) Time course of polymerization and (D–F) micrographs of rhodamine-phalloidin stained actin filaments from polymerization reactions: (D, • in A) actin alone at 1,000 s, or actin with (B, ▪ in A) 10 nM Arp2/3 complex with 900 nM GST-Myo1p-23A at 200 s, (C, □ in A) 10 nM Arp2/3 complex with 900 nM GST-Myo1p-23A and 500 nM Vrp1p-His at 60 s, (E, ▴ in A) 10 nM Arp2/3 complex with 500 nM GST-Wsp1p-VCA at 150 s, (F, ▵ in A) 10 nM Arp2/3 complex with 500 nM GST-N-WASP-VVCA at 80 s. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast

    doi: 10.1083/jcb.200502053

    Figure Lengend Snippet: Fluorescence microscopy of the products of actin polymerization reactions. Conditions: 4 μM monomeric actin (5% pyrene), 10 mM imidazole, pH 7.0, 50 mM KCl, 5 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN 3 , and 20 μM CaCl 2 . After mixing the reactants, the samples were split into two: (A) pyrene fluorescence was used to monitor the polymerization of one aliquot; (B–F) 4 μM rhodamine-phalloidin was added to the other aliquot. When each reaction was 80% complete, the aliquot with rhodamine-phalloidin was diluted 400 times into microscopy buffer (10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl 2 , 100 mM DTT, 20 μg/ml catalase, 100 μg/ml glucose oxidase, 3 mg/ml glucose, and 0.5% methylcellulose) and 2 μl were applied to the nitrocellulose-coated 22 × 22-mm coverslip. (A) Time course of polymerization and (D–F) micrographs of rhodamine-phalloidin stained actin filaments from polymerization reactions: (D, • in A) actin alone at 1,000 s, or actin with (B, ▪ in A) 10 nM Arp2/3 complex with 900 nM GST-Myo1p-23A at 200 s, (C, □ in A) 10 nM Arp2/3 complex with 900 nM GST-Myo1p-23A and 500 nM Vrp1p-His at 60 s, (E, ▴ in A) 10 nM Arp2/3 complex with 500 nM GST-Wsp1p-VCA at 150 s, (F, ▵ in A) 10 nM Arp2/3 complex with 500 nM GST-N-WASP-VVCA at 80 s. Bar, 10 μm.

    Article Snippet: Protein purification Native S. pombe Arp2/3 complex was purified from protease-deficient TM011 cells resuspended in buffer U (50 mM Hepes, pH 7.5, 100 mM KCl, 3 mM MgCl2 , 1 mM EGTA, 0.1 mM ATP, and 1 mM DTT) containing Complete (Roche) protease inhibitors and ruptured using a Microfluidizer (model M-110S; Microfluidics).

    Techniques: Fluorescence, Microscopy, Staining

    ASK1 expression is reduced in the presence of overexpressed CHIP. COS-7 cells were transiently transfected with HA-ASK1 with or without CHIP (wild type and H260Q). Twenty-four hours after transfection, cells were lysed in radioimmune precipitation

    Journal: Cell Stress & Chaperones

    Article Title: C-terminus of heat shock protein 70- interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1- dependent apoptosis

    doi: 10.1379/CSC-90R.1

    Figure Lengend Snippet: ASK1 expression is reduced in the presence of overexpressed CHIP. COS-7 cells were transiently transfected with HA-ASK1 with or without CHIP (wild type and H260Q). Twenty-four hours after transfection, cells were lysed in radioimmune precipitation

    Article Snippet: Transfected cells were harvested 24 hours after transfection and were lysed with radioimmune precipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM ethylene glycol tetra acetic acid (EGTA), 0.25% sodium deoxycholate, 1% Nonidet P-40, I mM Na3 VO4 , 50 mM NaF) supplemented with protease inhibitor cocktail (Roche Molecular Biochemicals) and 1 mM phenylmethylsulfonyl fluoride (PMSF).

    Techniques: Expressing, Chromatin Immunoprecipitation, Transfection