ethylenediaminetetraacetic acid etda disodium salt  (Millipore)


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  • 90
    Name:
    Ethylenediaminetetraacetic acid copper II disodium salt
    Description:

    Catalog Number:
    03668
    Price:
    None
    Applications:
    Used to eliminate inhibition of enzyme catalyzed reactions due to traces of heavy metals
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    Structured Review

    Millipore ethylenediaminetetraacetic acid etda disodium salt
    Ethylenediaminetetraacetic acid copper II disodium salt

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    Average 90 stars, based on 1 article reviews
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    High Performance Liquid Chromatography:

    Article Title: A 4-Oxo-2(E)-Nonenal-Derived Glutathione-Adduct from 15-Lipoxygenase-1-Mediated Oxidation of Cytosolic and Esterified Arachidonic Acid §
    Article Snippet: .. High performance liquid chromatography grade water, acetonitrile, hexane, methanol and isopropanol were obtained from Fisher Scientific Co. (Fair Lawn, NJ). glutathione, fetal bovine serum, calcium ionophore A23187, formic acid, trifluoroacetic acid, sodium borohydride, ethylenediaminetetraacetic acid (ETDA) disodium salt, diisopropylethylamine and 2,3,4,5,6-pentafluorobenzyl bromide were acquired from SigmaAldrich (St. Louis, MO). ..

    Modification:

    Article Title: A 4-Oxo-2(E)-Nonenal-Derived Glutathione-Adduct from 15-Lipoxygenase-1-Mediated Oxidation of Cytosolic and Esterified Arachidonic Acid §
    Article Snippet: Dulbecco's Modified Eagle's Medium (DMEM), phosphate buffer saline, Lipofectamine 2000 and geneticine were supplied by Invitrogen (Carlsbad, CA). .. High performance liquid chromatography grade water, acetonitrile, hexane, methanol and isopropanol were obtained from Fisher Scientific Co. (Fair Lawn, NJ). glutathione, fetal bovine serum, calcium ionophore A23187, formic acid, trifluoroacetic acid, sodium borohydride, ethylenediaminetetraacetic acid (ETDA) disodium salt, diisopropylethylamine and 2,3,4,5,6-pentafluorobenzyl bromide were acquired from SigmaAldrich (St. Louis, MO).

    BIA-KA:

    Article Title: A 4-Oxo-2(E)-Nonenal-Derived Glutathione-Adduct from 15-Lipoxygenase-1-Mediated Oxidation of Cytosolic and Esterified Arachidonic Acid §
    Article Snippet: The bicinchoninic acid (BCA) assay kit was obtained from Pierce (Rockford, IL). .. High performance liquid chromatography grade water, acetonitrile, hexane, methanol and isopropanol were obtained from Fisher Scientific Co. (Fair Lawn, NJ). glutathione, fetal bovine serum, calcium ionophore A23187, formic acid, trifluoroacetic acid, sodium borohydride, ethylenediaminetetraacetic acid (ETDA) disodium salt, diisopropylethylamine and 2,3,4,5,6-pentafluorobenzyl bromide were acquired from SigmaAldrich (St. Louis, MO).

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  • 95
    Millipore nmr buffer
    The m 6 A modification has minor effects on RREIIB structure and dynamics. (A) Secondary structure of RRE2Bm 6A68 , with the residues showing line-broadening with m 6 A, highlighted in red (with Mg 2+ ) and blue (no Mg 2+ ). Comparison of the 1D 1 H <t>NMR</t> spectrum of RREIIB m6A68 with or without Mg 2+ with the methyl peak indicated by arrows. The comparison of 1D imino spectra (B) and 2D [ 1 H, 13 C]-HSQC spectra (C) of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . Resonances exhibiting shifting are indicated using arrows, and those with ambiguous assignments denoted using an asterisk. (D) Normalized resonance intensities in 2D [ 1 H, 13 C]-HSQC spectra of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . A52-C8H8, A52-C2H2 and U56-C6H6 were used as a reference and normalized to 0.1. The sample conditions were 1.2–1.5 mM RREIIB m6A68 or RREIIB in 15 mM sodium phosphate, 25 mM <t>NaCl,</t> 0.1 mM EDTA, pH 6.4 with or without 3 mM MgCl 2 .
    Nmr Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore calpain reaction buffer
    Schematic diagram showing the relationship of <t>calpain/NF-κB/inflammation/NVU</t> damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
    Calpain Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore uch buffer
    Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with <t>DLB</t> (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by <t>UCH-L1</t> in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.
    Uch Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore jak2 ip buffer
    Erythropoietin Receptor-Mediated Activation of the <t>JAK2/STAT5,</t> RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).
    Jak2 Ip Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The m 6 A modification has minor effects on RREIIB structure and dynamics. (A) Secondary structure of RRE2Bm 6A68 , with the residues showing line-broadening with m 6 A, highlighted in red (with Mg 2+ ) and blue (no Mg 2+ ). Comparison of the 1D 1 H NMR spectrum of RREIIB m6A68 with or without Mg 2+ with the methyl peak indicated by arrows. The comparison of 1D imino spectra (B) and 2D [ 1 H, 13 C]-HSQC spectra (C) of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . Resonances exhibiting shifting are indicated using arrows, and those with ambiguous assignments denoted using an asterisk. (D) Normalized resonance intensities in 2D [ 1 H, 13 C]-HSQC spectra of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . A52-C8H8, A52-C2H2 and U56-C6H6 were used as a reference and normalized to 0.1. The sample conditions were 1.2–1.5 mM RREIIB m6A68 or RREIIB in 15 mM sodium phosphate, 25 mM NaCl, 0.1 mM EDTA, pH 6.4 with or without 3 mM MgCl 2 .

    Journal: PLoS ONE

    Article Title: m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB

    doi: 10.1371/journal.pone.0224850

    Figure Lengend Snippet: The m 6 A modification has minor effects on RREIIB structure and dynamics. (A) Secondary structure of RRE2Bm 6A68 , with the residues showing line-broadening with m 6 A, highlighted in red (with Mg 2+ ) and blue (no Mg 2+ ). Comparison of the 1D 1 H NMR spectrum of RREIIB m6A68 with or without Mg 2+ with the methyl peak indicated by arrows. The comparison of 1D imino spectra (B) and 2D [ 1 H, 13 C]-HSQC spectra (C) of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . Resonances exhibiting shifting are indicated using arrows, and those with ambiguous assignments denoted using an asterisk. (D) Normalized resonance intensities in 2D [ 1 H, 13 C]-HSQC spectra of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . A52-C8H8, A52-C2H2 and U56-C6H6 were used as a reference and normalized to 0.1. The sample conditions were 1.2–1.5 mM RREIIB m6A68 or RREIIB in 15 mM sodium phosphate, 25 mM NaCl, 0.1 mM EDTA, pH 6.4 with or without 3 mM MgCl 2 .

    Article Snippet: After measuring the concentration, the RNA samples were buffer-exchanged into NMR buffer (15 mM sodium phosphate, 25 mM NaCl, 0.1 mM EDTA, with or without 3 mM MgCl2 at pH = 6.4) three times using 3kDa Amicon Ultra centrifugal filters (EMD Millipore).

    Techniques: Modification, Nuclear Magnetic Resonance

    Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Mouse Assay, Activation Assay

    MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Activity Assay, Expressing, Western Blot

    Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.

    Journal: The American Journal of Pathology

    Article Title: Ubiquitination of ?-Synuclein Is Not Required for Formation of Pathological Inclusions in ?-Synucleinopathies

    doi:

    Figure Lengend Snippet: Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.

    Article Snippet: SDS-soluble fractions from the cingulate cortex of neuropathologically normal (NL) or DLB brains were diluted 40-fold in UCH buffer (50 mmol/L HEPES, pH 7.8, 0.5 mmol/L EDTA, 1 mmol/L dithiothreitol) and then concentrated 40-fold using a MicroconYM-10 (Millipore Corp., Bedford, MA) to remove SDS.

    Techniques: Western Blot, Recombinant, Immunoprecipitation, Isolation, Modification, In Vitro

    Erythropoietin Receptor-Mediated Activation of the JAK2/STAT5, RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).

    Journal: Cancer discovery

    Article Title: MEK-Dependent Negative Feedback Underlies BCR-ABL-Mediated Oncogene Addiction

    doi: 10.1158/2159-8290.CD-13-0235

    Figure Lengend Snippet: Erythropoietin Receptor-Mediated Activation of the JAK2/STAT5, RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).

    Article Snippet: Cells were lysed in JAK2 IP buffer (50mM Tris pH 7.6, 100mM NaCl, 1mM EDTA, 1mM EGTA, 0.5% NP40, 0.1% Triton) or RAS IP buffer (50mM Tris pH 7.5, 125mM NaCl, 6.5mM MgCl2 , 5% glycerol, 0.2% NP40) supplemented with 1% protease and 1% phosphatase inhibitors (Calbiochem).

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

    Global Gene Expression Analysis of Dasatinib Treated K562 Cells Identifies Candidate Mediators Responsible for the Attenuation of Myeloid GF-R Signaling in CML Cells A. ). The heat map at the bottom highlights a few of the genes with increased expression following dasatinib treatment in K562 cells. B. Quantitative PCR (qPCR) analysis of potential negative feedback genes in K562 cells treated with dasatinib (100nM), imatinib (1μM), or PD0325901 (500nM). The average fold expression change (2 (−ΔΔCt) ) and standard deviation (SD fold-change ) for each gene is represented (n=3). C. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of RAS and ERK activity before and after hEPO-stimulation (10′) in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 500nM PD0325901, or dasatinib/PD0325901. RAS activity was monitored using a RAS-GTP pulldown assay. D. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of JAK2 and STAT5 activity before and after hEPO-stimulation in K562 cells treated under the same experimental conditions as (A). JAK2 activation was determined by immunoprecipitation and activation loop (Y1007) phosphorylation.

    Journal: Cancer discovery

    Article Title: MEK-Dependent Negative Feedback Underlies BCR-ABL-Mediated Oncogene Addiction

    doi: 10.1158/2159-8290.CD-13-0235

    Figure Lengend Snippet: Global Gene Expression Analysis of Dasatinib Treated K562 Cells Identifies Candidate Mediators Responsible for the Attenuation of Myeloid GF-R Signaling in CML Cells A. ). The heat map at the bottom highlights a few of the genes with increased expression following dasatinib treatment in K562 cells. B. Quantitative PCR (qPCR) analysis of potential negative feedback genes in K562 cells treated with dasatinib (100nM), imatinib (1μM), or PD0325901 (500nM). The average fold expression change (2 (−ΔΔCt) ) and standard deviation (SD fold-change ) for each gene is represented (n=3). C. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of RAS and ERK activity before and after hEPO-stimulation (10′) in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 500nM PD0325901, or dasatinib/PD0325901. RAS activity was monitored using a RAS-GTP pulldown assay. D. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of JAK2 and STAT5 activity before and after hEPO-stimulation in K562 cells treated under the same experimental conditions as (A). JAK2 activation was determined by immunoprecipitation and activation loop (Y1007) phosphorylation.

    Article Snippet: Cells were lysed in JAK2 IP buffer (50mM Tris pH 7.6, 100mM NaCl, 1mM EDTA, 1mM EGTA, 0.5% NP40, 0.1% Triton) or RAS IP buffer (50mM Tris pH 7.5, 125mM NaCl, 6.5mM MgCl2 , 5% glycerol, 0.2% NP40) supplemented with 1% protease and 1% phosphatase inhibitors (Calbiochem).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Activity Assay, Activation Assay, Immunoprecipitation