ethylenediaminetetraacetic acid edta  (Millipore)

 
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    Name:
    Ethylenediaminetetraacetic acid
    Description:
    Ethylenediaminetetraacetic acid EDTA is a hydrophilic metal chelating agent that transforms metal ions into inactive cyclic metal complexes Hence it has industrial application for resolving metal contamination
    Catalog Number:
    E9884
    Price:
    None
    Applications:
    Ethylenediaminetetraacetic acid has been used: . as a component of labeling buffer, which is used in the washing step during cell fractionation{155) . for investigating the effect of papain enzyme on wound debridement and other skin issues . for harvesting rat arterial smooth muscle cell after its pre-treatment for its use in flow cytometry analysis
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    Structured Review

    Millipore ethylenediaminetetraacetic acid edta
    Ethylenediaminetetraacetic acid
    Ethylenediaminetetraacetic acid EDTA is a hydrophilic metal chelating agent that transforms metal ions into inactive cyclic metal complexes Hence it has industrial application for resolving metal contamination
    https://www.bioz.com/result/ethylenediaminetetraacetic acid edta/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ethylenediaminetetraacetic acid edta - by Bioz Stars, 2021-04
    97/100 stars

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    Related Articles

    Construct:

    Article Title: Combined decellularisation and dehydration improves the mechanical properties of tissue-engineered sinews
    Article Snippet: An individual sinew construct after 3 weeks in culture is displayed in . .. Decellularisation After 3 weeks of culture, sinew constructs were removed from culture and decellularised using an established decellularisation protocol., Briefly, constructs were soaked in 0.1 wt% ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich) in deionised water for 4 h at room temperature. .. Next, the constructs were washed in 0.1 wt% sodium dodecyl sulphate (SDS; Sigma-Aldrich) in 0.1% EDTA for 24 h with a single change of solution at 12 h. The constructs were then washed for 1 h in phosphate-buffered saline (PBS) with a single change of PBS after 30 min. Constructs were stored in PBS until tensile testing.

    Western Blot:

    Article Title: The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia
    Article Snippet: .. Western blot and immunoprecipitation Total cell extraction was performed using co-immunoprecipitation buffer [150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 50 mM HEPES (pH, 7.5), 1% Triton and 10% glycerol] supplemented with protease inhibitor (cat no. 036K4082; Sigma-Aldrich) and a phosphatase cocktail composed of PMSF and Na3 VO4 . .. Following quantification by Bio-Rad Protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 30 µg protein extract was denatured, reduced, separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes.

    Immunoprecipitation:

    Article Title: The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia
    Article Snippet: .. Western blot and immunoprecipitation Total cell extraction was performed using co-immunoprecipitation buffer [150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 50 mM HEPES (pH, 7.5), 1% Triton and 10% glycerol] supplemented with protease inhibitor (cat no. 036K4082; Sigma-Aldrich) and a phosphatase cocktail composed of PMSF and Na3 VO4 . .. Following quantification by Bio-Rad Protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 30 µg protein extract was denatured, reduced, separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes.

    Protease Inhibitor:

    Article Title: The targetable role of herpes virus-associated ubiquitin-specific protease (HAUSP) in p190 BCR-ABL leukemia
    Article Snippet: .. Western blot and immunoprecipitation Total cell extraction was performed using co-immunoprecipitation buffer [150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 50 mM HEPES (pH, 7.5), 1% Triton and 10% glycerol] supplemented with protease inhibitor (cat no. 036K4082; Sigma-Aldrich) and a phosphatase cocktail composed of PMSF and Na3 VO4 . .. Following quantification by Bio-Rad Protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 30 µg protein extract was denatured, reduced, separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes.

    Concentration Assay:

    Article Title: C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation
    Article Snippet: To detect low cytokine levels in cell culture supernatants, for IL-1β the Human IL-1 beta/IL-1F2 DuoSet ELISA (R & D Systems) was used, whereas IL6 and TNF-α were measured by the Human Quantikine® Immunoassays (R & D Systems, Minneapolis, MN, USA). .. Endogenous CRP was dissolved at a concentration of 5 µg/ml in PBS devoid of Ca2+ and Mg2+ (Gibco) containing 1.1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich), incubated at 37°C for 15 min followed by ultrafiltration using Amicon® Ultra centrifugal filters. .. The high molecular weight fraction was diluted in PBS/EDTA, ultrafiltrated, and transferred to PBS, 5 mM Ca2+ , without EDTA by two additional ultrafiltration steps.

    Incubation:

    Article Title: C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation
    Article Snippet: To detect low cytokine levels in cell culture supernatants, for IL-1β the Human IL-1 beta/IL-1F2 DuoSet ELISA (R & D Systems) was used, whereas IL6 and TNF-α were measured by the Human Quantikine® Immunoassays (R & D Systems, Minneapolis, MN, USA). .. Endogenous CRP was dissolved at a concentration of 5 µg/ml in PBS devoid of Ca2+ and Mg2+ (Gibco) containing 1.1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich), incubated at 37°C for 15 min followed by ultrafiltration using Amicon® Ultra centrifugal filters. .. The high molecular weight fraction was diluted in PBS/EDTA, ultrafiltrated, and transferred to PBS, 5 mM Ca2+ , without EDTA by two additional ultrafiltration steps.

    High Performance Liquid Chromatography:

    Article Title: An Interaction of Rhamnolipids with Cu2+ Ions
    Article Snippet: .. The rhamnolipid R-95 (the 1:1 mixture of mono- and dirhamnolipids with the HPLC purity; the mean molecular weight of 577 g mol−1 , p K a = 5.6), methylglycinediacetic acid (MGDA; analytical grade, p K a = 14.6 [ ]), ethylenediaminetetraacetic acid (EDTA; analytical grade), potassium chloride (KCl), copper (II) sulfate (CuSO4 ; analytical grade), potassium hydroxide (KOH; analytical grade), and sulfuric acid (H2 SO4 ) were purchased from Sigma Aldrich (Poznan, Poland) company. .. Distilled filtrated (0.2 µm Whatman filters; GE Healthcare UK Ltd., Little Chalfont, UK) water with the electrolytic conductivity of 0.001 ms cm–1 at 20 ± 0.1 °C) was used for the solutions preparation.

    Molecular Weight:

    Article Title: An Interaction of Rhamnolipids with Cu2+ Ions
    Article Snippet: .. The rhamnolipid R-95 (the 1:1 mixture of mono- and dirhamnolipids with the HPLC purity; the mean molecular weight of 577 g mol−1 , p K a = 5.6), methylglycinediacetic acid (MGDA; analytical grade, p K a = 14.6 [ ]), ethylenediaminetetraacetic acid (EDTA; analytical grade), potassium chloride (KCl), copper (II) sulfate (CuSO4 ; analytical grade), potassium hydroxide (KOH; analytical grade), and sulfuric acid (H2 SO4 ) were purchased from Sigma Aldrich (Poznan, Poland) company. .. Distilled filtrated (0.2 µm Whatman filters; GE Healthcare UK Ltd., Little Chalfont, UK) water with the electrolytic conductivity of 0.001 ms cm–1 at 20 ± 0.1 °C) was used for the solutions preparation.

    Flow Cytometry:

    Article Title: Monocytes mediate Salmonella Typhimurium-induced tumour growth inhibition
    Article Snippet: A portion of the cells were counted using Trypan Blue exclusion dye (Sigma, T8154). .. Cells were resuspended in flow cytometry buffer (FB: 2% FCS, 3 nM EDTA (Sigma, E9884) in PBS). .. Cells were first stained in Fixable Viability Dye eFluor® 780 (eBioscience) in PBS for 15-20 min on ice, in the dark.

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    Millipore edta
    Confirmation of rhGH protein expression in the pig milk and schematics of pretreatment procedures. (A) Confirmation of rhGH protein expression in the pig milk by Western blot assay. Protein samples were loaded onto 13.5% SDS-PAGE gels and analyzed by Western blotting. Then, the blots were stained with Coomassie brilliant blue (CBB). M: marker, C: hGH standard (Ministry of Food and Drug Safety, Korea) 100 μg, 1: rhGH <t>transgenic</t> pig; identification number 68–1 (hGH-LYY 17, F0 generation), 2: rhGH transgenic pig; identification number 94–2 (hGH-LYY 27, F0 generation), d: lactating day, sample loading: crude milk 1 μL, H.C.: Heavy chain, L.C.: Light chain. (B) Schematic of milk pretreatment procedures. (C) CBB staining and Western blotting in different steps of rhGH transgenic porcine milk pretreatment. Protein samples were separated using a 12% SDS-PAGE gel. 1; crude milk, 2; 20 mM <t>EDTA</t> treatment supernatant, 3; 2 M Tris pH 7.4 treatment; 0.2-μm filter permeate, 5; 300-kDa filter permeate, 6; 5-kDa filter retentate. *: impurity (porcine beta- lactoglobulin), **: rhGH.
    Edta, supplied by Millipore, 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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    edta - by Bioz Stars, 2021-04
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    97
    Millipore co immunoprecipitation buffer
    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 <t>co-immunoprecipitation</t> assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.
    Co Immunoprecipitation Buffer, supplied by Millipore, 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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    99
    Millipore gst lysis buffer
    (A) Endogenous MYCN and <t>p53</t> co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro <t>GST-C-MYC</t> pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.
    Gst Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Confirmation of rhGH protein expression in the pig milk and schematics of pretreatment procedures. (A) Confirmation of rhGH protein expression in the pig milk by Western blot assay. Protein samples were loaded onto 13.5% SDS-PAGE gels and analyzed by Western blotting. Then, the blots were stained with Coomassie brilliant blue (CBB). M: marker, C: hGH standard (Ministry of Food and Drug Safety, Korea) 100 μg, 1: rhGH transgenic pig; identification number 68–1 (hGH-LYY 17, F0 generation), 2: rhGH transgenic pig; identification number 94–2 (hGH-LYY 27, F0 generation), d: lactating day, sample loading: crude milk 1 μL, H.C.: Heavy chain, L.C.: Light chain. (B) Schematic of milk pretreatment procedures. (C) CBB staining and Western blotting in different steps of rhGH transgenic porcine milk pretreatment. Protein samples were separated using a 12% SDS-PAGE gel. 1; crude milk, 2; 20 mM EDTA treatment supernatant, 3; 2 M Tris pH 7.4 treatment; 0.2-μm filter permeate, 5; 300-kDa filter permeate, 6; 5-kDa filter retentate. *: impurity (porcine beta- lactoglobulin), **: rhGH.

    Journal: PLoS ONE

    Article Title: Structural and functional characterization of recombinant human growth hormone isolated from transgenic pig milk

    doi: 10.1371/journal.pone.0236788

    Figure Lengend Snippet: Confirmation of rhGH protein expression in the pig milk and schematics of pretreatment procedures. (A) Confirmation of rhGH protein expression in the pig milk by Western blot assay. Protein samples were loaded onto 13.5% SDS-PAGE gels and analyzed by Western blotting. Then, the blots were stained with Coomassie brilliant blue (CBB). M: marker, C: hGH standard (Ministry of Food and Drug Safety, Korea) 100 μg, 1: rhGH transgenic pig; identification number 68–1 (hGH-LYY 17, F0 generation), 2: rhGH transgenic pig; identification number 94–2 (hGH-LYY 27, F0 generation), d: lactating day, sample loading: crude milk 1 μL, H.C.: Heavy chain, L.C.: Light chain. (B) Schematic of milk pretreatment procedures. (C) CBB staining and Western blotting in different steps of rhGH transgenic porcine milk pretreatment. Protein samples were separated using a 12% SDS-PAGE gel. 1; crude milk, 2; 20 mM EDTA treatment supernatant, 3; 2 M Tris pH 7.4 treatment; 0.2-μm filter permeate, 5; 300-kDa filter permeate, 6; 5-kDa filter retentate. *: impurity (porcine beta- lactoglobulin), **: rhGH.

    Article Snippet: Pretreatment of transgenic sow milk (sample preparation) The transgenic milk samples were diluted with a 2-fold volume of EDTA (Sigma, St. Louis, MO, USA) at a final concentration of 20 mM to extract rhGH within casein micelles.

    Techniques: Expressing, Western Blot, SDS Page, Staining, Marker, Transgenic Assay

    Effect of protease inhibitors on antimicrobial activities of MP-V1 in the increased Salmonella population density. The effect of a protease inhibitor cocktail (Sigma-Aldrich, Milwaukee, WI, USA) and its components (23 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 2 mM bestatin, 0.3 mM pepstatin A, 0.3 mM E-64 and 100 mM EDTA) on antimicrobial activities was examined using 0.5 to 10 μL doses against 10 8 cfu/mL of Salmonella Enteritidis ( A ); Salmonella Gallinarum ( B ); and Salmonella Typhimurium ( C ). The MP-V1 was used at the MICs determined in 10 6 cfu/mL. Data are means ± SE ( n = 3). Different letters indicate significant differences by the one-way ANOVA/Duncan ( p

    Journal: Toxins

    Article Title: Anti-Salmonella Activity Modulation of Mastoparan V1—A Wasp Venom Toxin—Using Protease Inhibitors, and Its Efficient Production via an Escherichia coli Secretion System

    doi: 10.3390/toxins9100321

    Figure Lengend Snippet: Effect of protease inhibitors on antimicrobial activities of MP-V1 in the increased Salmonella population density. The effect of a protease inhibitor cocktail (Sigma-Aldrich, Milwaukee, WI, USA) and its components (23 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 2 mM bestatin, 0.3 mM pepstatin A, 0.3 mM E-64 and 100 mM EDTA) on antimicrobial activities was examined using 0.5 to 10 μL doses against 10 8 cfu/mL of Salmonella Enteritidis ( A ); Salmonella Gallinarum ( B ); and Salmonella Typhimurium ( C ). The MP-V1 was used at the MICs determined in 10 6 cfu/mL. Data are means ± SE ( n = 3). Different letters indicate significant differences by the one-way ANOVA/Duncan ( p

    Article Snippet: The protease inhibitor cocktail for bacterial use and the protease inhibitors, including AEBSF, bestatin, pepstatin A, E-64 and EDTA, were purchased from Sigma-Aldrich (Milwaukee, WI, USA).

    Techniques: Protease Inhibitor

    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Immunoprecipitation, Western Blot, Functional Assay

    AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Chemotaxis Assay, Sequencing, Functional Assay, Binding Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

    (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Article Snippet: GST-p53 cells were lysed in GST lysis buffer (1% Triton, 1 μg/μl lysozyme, 0.5 mM EDTA, and 1 mM PMSF in phosphate buffered saline), purified and immobilized on glutathione-agarose beads (Sigma Aldrich).

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Western Blot, In Vitro, Expressing, Incubation, Staining, Purification, Recombinant, Transfection, Cell Culture, Amplification

    (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Article Snippet: GST-p53 cells were lysed in GST lysis buffer (1% Triton, 1 μg/μl lysozyme, 0.5 mM EDTA, and 1 mM PMSF in phosphate buffered saline), purified and immobilized on glutathione-agarose beads (Sigma Aldrich).

    Techniques: Construct, Activation Assay, Binding Assay, Clone Assay, Plasmid Preparation, Purification, Amplification, Incubation, Negative Control, Staining, Pull Down Assay, Transfection