gst lysis buffer  (Millipore)

 
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    Name:
    Lysozyme
    Description:
    Muramidase Mucopeptide N acetylmuramoyl hydrolase
    Catalog Number:
    10837059001
    Price:
    None
    Applications:
    Lysozyme has been used for:. Cell wall degradation. Preparation of protoplasts. Bacteriolysis. Pharmacology (anti-inflammatory effect on mucous membrane; healing of tissue; protection against bleeding; normalization of mucus secretion; dissolution of pyrogens; potentiation of antibiotics). Food and drinks (flavor enhancer). Sample preparation prior to isolation of nucleic acids
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    Structured Review

    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.
    Muramidase Mucopeptide N acetylmuramoyl hydrolase
    https://www.bioz.com/result/gst lysis buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gst lysis buffer - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma"

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24859

    (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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    2) Product Images from "MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma"

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24859

    (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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

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    Generated:

    Article Title: ASK1 Negatively Regulates the 26 S Proteasome *
    Article Snippet: Proteasome activity was assayed using ubiquitin-conjugated lysozyme as a substrate. .. Ubiquitin-conjugated lysozyme was generated by ubiquitinating lysozyme in vitro , using a ubiquitin protein-conjugation kit (Calbiochem). ..

    In Vitro:

    Article Title: ASK1 Negatively Regulates the 26 S Proteasome *
    Article Snippet: Proteasome activity was assayed using ubiquitin-conjugated lysozyme as a substrate. .. Ubiquitin-conjugated lysozyme was generated by ubiquitinating lysozyme in vitro , using a ubiquitin protein-conjugation kit (Calbiochem). ..

    SDS Page:

    Article Title: Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins
    Article Snippet: Calibration was performed using an authentic standard of myoglobin (Waters, Milford, MA). .. HNE-treated lysozyme and BSA, as well as their untreated (control) counterparts, were subjected to SDS-PAGE, then stained with Coomassie Blue; the gel bands were excised, subjected to in-gel trypsin digestion, and the resultant peptides extracted and purified (C18 Ziptip™; Millipore, Billerica, MA), following by nanoLC-MS/MS analysis using a nanoACQUITY® UPLC coupled with a QTOF Ultima API mass spectrometer (Waters, Milford, MA), as previously described [ ]. shows a schematic outline of the direct infusion experiment ( ) and the nanoLC-MS/MS experiment , respectively. .. Data analysis was performed using ProteinLynx Global SERVER (PLGS version 2.4, Waters), with a custom-created database against unmodified and modified lysozyme and BSA, as well as using de novo ).

    Staining:

    Article Title: Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins
    Article Snippet: Calibration was performed using an authentic standard of myoglobin (Waters, Milford, MA). .. HNE-treated lysozyme and BSA, as well as their untreated (control) counterparts, were subjected to SDS-PAGE, then stained with Coomassie Blue; the gel bands were excised, subjected to in-gel trypsin digestion, and the resultant peptides extracted and purified (C18 Ziptip™; Millipore, Billerica, MA), following by nanoLC-MS/MS analysis using a nanoACQUITY® UPLC coupled with a QTOF Ultima API mass spectrometer (Waters, Milford, MA), as previously described [ ]. shows a schematic outline of the direct infusion experiment ( ) and the nanoLC-MS/MS experiment , respectively. .. Data analysis was performed using ProteinLynx Global SERVER (PLGS version 2.4, Waters), with a custom-created database against unmodified and modified lysozyme and BSA, as well as using de novo ).

    Purification:

    Article Title: Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins
    Article Snippet: Calibration was performed using an authentic standard of myoglobin (Waters, Milford, MA). .. HNE-treated lysozyme and BSA, as well as their untreated (control) counterparts, were subjected to SDS-PAGE, then stained with Coomassie Blue; the gel bands were excised, subjected to in-gel trypsin digestion, and the resultant peptides extracted and purified (C18 Ziptip™; Millipore, Billerica, MA), following by nanoLC-MS/MS analysis using a nanoACQUITY® UPLC coupled with a QTOF Ultima API mass spectrometer (Waters, Milford, MA), as previously described [ ]. shows a schematic outline of the direct infusion experiment ( ) and the nanoLC-MS/MS experiment , respectively. .. Data analysis was performed using ProteinLynx Global SERVER (PLGS version 2.4, Waters), with a custom-created database against unmodified and modified lysozyme and BSA, as well as using de novo ).

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma
    Article Snippet: BL21 E. coli were transformed with either MYCN-6×His or GST-p53 vector and protein expression was induced with 0.4 mM IPTG for 8 h at 25°C. .. 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). .. BL-21 E.Coli cells expressing MYCN-6×His were lysed in His lysis buffer (50 mM NaH2PO4, 1% Triton, 1 μg/μl lysozyme, 1 mM PMSF, 300 mM NaCl, and 20 mM imidazole), purified using HIS-Select Nickel Affinity Gel (Sigma Aldrich) and eluted with 350 mM imidazole in His lysis buffer without lysozyme.

    Mass Spectrometry:

    Article Title: Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins
    Article Snippet: Calibration was performed using an authentic standard of myoglobin (Waters, Milford, MA). .. HNE-treated lysozyme and BSA, as well as their untreated (control) counterparts, were subjected to SDS-PAGE, then stained with Coomassie Blue; the gel bands were excised, subjected to in-gel trypsin digestion, and the resultant peptides extracted and purified (C18 Ziptip™; Millipore, Billerica, MA), following by nanoLC-MS/MS analysis using a nanoACQUITY® UPLC coupled with a QTOF Ultima API mass spectrometer (Waters, Milford, MA), as previously described [ ]. shows a schematic outline of the direct infusion experiment ( ) and the nanoLC-MS/MS experiment , respectively. .. Data analysis was performed using ProteinLynx Global SERVER (PLGS version 2.4, Waters), with a custom-created database against unmodified and modified lysozyme and BSA, as well as using de novo ).

    Amplification:

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    Polymerase Chain Reaction:

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    Clone Assay:

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    Drug Susceptibility Assay:

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    Immunoprecipitation:

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    Lysis:

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    Article Snippet: .. For immunoprecipitation of FtsA, a cell pellet was thawed and resuspended in 100 μl of FtsA lysis buffer (immunoprecipitation wash buffer, 4 mg ml−1 lysozyme (Sigma-Aldrich), protease inhibitor cocktail (Complete Mini, EDTA-free protease inhibitor cocktail tablet, Roche) and placed on ice for 30 min. .. Cell lysate from each sample was then resuspended in 0.9 ml of immunoprecipitation wash buffer (50 mM Tris-HCl pH 8.2, 450 mM NaCl and 1% Triton X-100).

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma
    Article Snippet: BL21 E. coli were transformed with either MYCN-6×His or GST-p53 vector and protein expression was induced with 0.4 mM IPTG for 8 h at 25°C. .. 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). .. BL-21 E.Coli cells expressing MYCN-6×His were lysed in His lysis buffer (50 mM NaH2PO4, 1% Triton, 1 μg/μl lysozyme, 1 mM PMSF, 300 mM NaCl, and 20 mM imidazole), purified using HIS-Select Nickel Affinity Gel (Sigma Aldrich) and eluted with 350 mM imidazole in His lysis buffer without lysozyme.

    Protease Inhibitor:

    Article Title: ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division
    Article Snippet: .. For immunoprecipitation of FtsA, a cell pellet was thawed and resuspended in 100 μl of FtsA lysis buffer (immunoprecipitation wash buffer, 4 mg ml−1 lysozyme (Sigma-Aldrich), protease inhibitor cocktail (Complete Mini, EDTA-free protease inhibitor cocktail tablet, Roche) and placed on ice for 30 min. .. Cell lysate from each sample was then resuspended in 0.9 ml of immunoprecipitation wash buffer (50 mM Tris-HCl pH 8.2, 450 mM NaCl and 1% Triton X-100).

    Incubation:

    Article Title: Airway Mucins Inhibit Oxidative and Non-Oxidative Bacterial Killing by Human Neutrophils
    Article Snippet: Antimicrobial Peptides/Proteins, Mucins, and Bacterial Killing Subcultures of PAO1 were adjusted to 2.5 × 105 bacteria/ml in 10 mM phosphate buffer (pH 7.4) and incubated with 0–4 mg/ml CFAM or salivary mucins for 20 min at RT. .. Each sample was then incubated for 2 h at 37°C with gentle agitation, either alone or with one of the following antimicrobial peptide/proteins preparations: 6 μg/ml of recombinant human neutrophil lysozyme; lysozyme and 0–5 mg/ml of chondroitin sulfate; 4 μg/ml of human β defensin-2 (HBD-2); or neutrophil granule extract containing 8 μg/ml of lysozyme (Sigma-Aldrich). .. The samples were then diluted in PBS, plated on Müller-Hinton agar, incubated overnight at 34°C, and CFU were counted by standard plate-counting procedures ( ; ).

    Recombinant:

    Article Title: Airway Mucins Inhibit Oxidative and Non-Oxidative Bacterial Killing by Human Neutrophils
    Article Snippet: Antimicrobial Peptides/Proteins, Mucins, and Bacterial Killing Subcultures of PAO1 were adjusted to 2.5 × 105 bacteria/ml in 10 mM phosphate buffer (pH 7.4) and incubated with 0–4 mg/ml CFAM or salivary mucins for 20 min at RT. .. Each sample was then incubated for 2 h at 37°C with gentle agitation, either alone or with one of the following antimicrobial peptide/proteins preparations: 6 μg/ml of recombinant human neutrophil lysozyme; lysozyme and 0–5 mg/ml of chondroitin sulfate; 4 μg/ml of human β defensin-2 (HBD-2); or neutrophil granule extract containing 8 μg/ml of lysozyme (Sigma-Aldrich). .. The samples were then diluted in PBS, plated on Müller-Hinton agar, incubated overnight at 34°C, and CFU were counted by standard plate-counting procedures ( ; ).

    Plasmid Preparation:

    Article Title: Metal ions‐binding T4 lysozyme as an intramolecular protein purification tag compatible with X‐ray crystallography
    Article Snippet: Our study provides a structural rationale and methodological insights for its utilization as an intramolecular protein purification tag for purification of recombinant proteins by the IMAC technique. .. Wild‐type phage T4 lysozyme was cloned into NdeI and KpnI restriction sites of the pRSFD vector (Novagen) using routine restriction cloning. .. Truncated and metal ions‐binding T4L mutants were generated using the Phusion Site‐Directed Mutagenesis Kit (Thermo Scientific).

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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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    99
    Millipore protease inhibitor cocktail iii
    The protein complementation assay confirms the effects of the R282H mutation and a phosphorylation site mutation on TIN2L interaction with TRF2. (A) Quantification of fluorescence from coexpression of V1-TRF2 with TIN2L-V2, TIN2L-D391K+D395K-V2, or TIN2L-R282H-V2. Error bars represent the <t>SDs</t> from <t>three</t> separate transfections each measured in triplicate. *, P
    Protease Inhibitor Cocktail Iii, 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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    97
    Millipore lysis buffer b gst dyrk1a fusion proteins
    Phosphorylation of Ser-520 on <t>DYRK1A</t> mediates its interaction with the protein 14-3-3β. (A) Lysates from HEK293 cells transiently transfected with pHA-DYRK1A were incubated for 30 min at 30°C in phosphatase buffer (lane 1) supplemented with alkaline phosphatase (lane 2) or with alkaline phosphatase and sodium pyrophosphate (lane 3) as indicated. After incubation with <t>GST-14-3-3β</t> immobilized on glutathione-Sepharose beads, bound protein was detected by immunoblotting (IB) with anti-HA antibody (top). An immunoblot of cell lysates representing 10% of the inputs is shown (bottom). (B) Schematic diagram of DYRK1A showing the domain structure: KINASE, kinase catalytic domain; PEST, PEST domain; His, histidine-repeat; and S/T, Ser/Thr-rich region. The sequence and the position of the putative 14-3-3 binding motif in DYRK1A are indicated and compared with a mode I 14-3-3 binding consensus sequence. The putative phosphorylated Ser residue is marked in bold. (C) HEK293 cells were transfected with pHA-DYRK1Awt and pHA-DYRK1A/S520A, and soluble lysates were incubated with unfused GST or GST-14-3-3β. Samples were analyzed by SDS-PAGE (10%) followed by immunoblotting with anti-HA antibody. Lysate represents 10% of the input. (D) The histogram presents data from three independent experiments in which the ratio bound/input in DYRK1Awt was arbitrarily set as 100. Data are expressed as a percentage of the mean value. Error bars represent SEM (standard error of the mean). (E) Subcellular localization of HA-DYRK1Awt and the mutant HA-DYRK1A/S520A, expressed in U2-OS cells, by indirect immunofluorescence analysis with anti-HA antibody followed by a FITC-conjugated anti-mouse secondary antibody.
    Lysis Buffer B Gst Dyrk1a Fusion Proteins, 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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    (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

    (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

    The protein complementation assay confirms the effects of the R282H mutation and a phosphorylation site mutation on TIN2L interaction with TRF2. (A) Quantification of fluorescence from coexpression of V1-TRF2 with TIN2L-V2, TIN2L-D391K+D395K-V2, or TIN2L-R282H-V2. Error bars represent the SDs from three separate transfections each measured in triplicate. *, P

    Journal: Molecular and Cellular Biology

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion

    doi: 10.1128/MCB.00025-18

    Figure Lengend Snippet: The protein complementation assay confirms the effects of the R282H mutation and a phosphorylation site mutation on TIN2L interaction with TRF2. (A) Quantification of fluorescence from coexpression of V1-TRF2 with TIN2L-V2, TIN2L-D391K+D395K-V2, or TIN2L-R282H-V2. Error bars represent the SDs from three separate transfections each measured in triplicate. *, P

    Article Snippet: Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water.

    Techniques: Mutagenesis, Fluorescence, Transfection

    Phosphorylation of Ser-520 on DYRK1A mediates its interaction with the protein 14-3-3β. (A) Lysates from HEK293 cells transiently transfected with pHA-DYRK1A were incubated for 30 min at 30°C in phosphatase buffer (lane 1) supplemented with alkaline phosphatase (lane 2) or with alkaline phosphatase and sodium pyrophosphate (lane 3) as indicated. After incubation with GST-14-3-3β immobilized on glutathione-Sepharose beads, bound protein was detected by immunoblotting (IB) with anti-HA antibody (top). An immunoblot of cell lysates representing 10% of the inputs is shown (bottom). (B) Schematic diagram of DYRK1A showing the domain structure: KINASE, kinase catalytic domain; PEST, PEST domain; His, histidine-repeat; and S/T, Ser/Thr-rich region. The sequence and the position of the putative 14-3-3 binding motif in DYRK1A are indicated and compared with a mode I 14-3-3 binding consensus sequence. The putative phosphorylated Ser residue is marked in bold. (C) HEK293 cells were transfected with pHA-DYRK1Awt and pHA-DYRK1A/S520A, and soluble lysates were incubated with unfused GST or GST-14-3-3β. Samples were analyzed by SDS-PAGE (10%) followed by immunoblotting with anti-HA antibody. Lysate represents 10% of the input. (D) The histogram presents data from three independent experiments in which the ratio bound/input in DYRK1Awt was arbitrarily set as 100. Data are expressed as a percentage of the mean value. Error bars represent SEM (standard error of the mean). (E) Subcellular localization of HA-DYRK1Awt and the mutant HA-DYRK1A/S520A, expressed in U2-OS cells, by indirect immunofluorescence analysis with anti-HA antibody followed by a FITC-conjugated anti-mouse secondary antibody.

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-08-0668

    Figure Lengend Snippet: Phosphorylation of Ser-520 on DYRK1A mediates its interaction with the protein 14-3-3β. (A) Lysates from HEK293 cells transiently transfected with pHA-DYRK1A were incubated for 30 min at 30°C in phosphatase buffer (lane 1) supplemented with alkaline phosphatase (lane 2) or with alkaline phosphatase and sodium pyrophosphate (lane 3) as indicated. After incubation with GST-14-3-3β immobilized on glutathione-Sepharose beads, bound protein was detected by immunoblotting (IB) with anti-HA antibody (top). An immunoblot of cell lysates representing 10% of the inputs is shown (bottom). (B) Schematic diagram of DYRK1A showing the domain structure: KINASE, kinase catalytic domain; PEST, PEST domain; His, histidine-repeat; and S/T, Ser/Thr-rich region. The sequence and the position of the putative 14-3-3 binding motif in DYRK1A are indicated and compared with a mode I 14-3-3 binding consensus sequence. The putative phosphorylated Ser residue is marked in bold. (C) HEK293 cells were transfected with pHA-DYRK1Awt and pHA-DYRK1A/S520A, and soluble lysates were incubated with unfused GST or GST-14-3-3β. Samples were analyzed by SDS-PAGE (10%) followed by immunoblotting with anti-HA antibody. Lysate represents 10% of the input. (D) The histogram presents data from three independent experiments in which the ratio bound/input in DYRK1Awt was arbitrarily set as 100. Data are expressed as a percentage of the mean value. Error bars represent SEM (standard error of the mean). (E) Subcellular localization of HA-DYRK1Awt and the mutant HA-DYRK1A/S520A, expressed in U2-OS cells, by indirect immunofluorescence analysis with anti-HA antibody followed by a FITC-conjugated anti-mouse secondary antibody.

    Article Snippet: Bacterial lysates were incubated with glutathione-Sepharose 4B beads (GE Healthcare) for 45 min at RT and washed four times with lysis buffer B. GST-DYRK1A fusion proteins were eluted with 10 mM reduced glutathione (Sigma-Aldrich) in 50 mM Tris-HCl, pH 8, and dialyzed against a buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM EDTA.

    Techniques: Transfection, Incubation, Sequencing, Binding Assay, SDS Page, Mutagenesis, Immunofluorescence

    DYRK1A autophosphorylates Ser-520 through an intramolecular reaction. (A) Bacterially expressed GST fusion proteins of wild-type DYRK1A (wt, lane 1) and the mutants DYRK1A/S520A (lane 2) and DYRK1A/K179R (lane 3) were immunoblotted with anti-DYRK1A pS520 (top), anti-phospho-Tyr (PY20; middle), and anti-GST (bottom) antibodies. The positions of marker proteins (in kilodaltons) are indicated. Arrows indicate full-length GST-fusion proteins, and asterisks indicate truncated GST-fusion proteins lacking the C-terminal (∼150) amino acids). The different patterns in DYRK1Awt, DYRK1AS520A, and DYRK1AK179R detected with the anti-GST antibody are due to differences in phosphorylation levels as shown in Supplemental Figure S3, A. (B) HA-tagged proteins corresponding to the two major isoforms of DYRK1A (lanes 1 and 2) and to distinct catalytically inactive point mutants (lanes 3–5) were overexpressed by transient transfection into U2-OS cells. Phosphorylation of Ser-520 was detected by immunoblotting with the phospho-specific anti-DYRK1A pS520 antibody (top). To control for equal expression of the fusion proteins, membranes were stripped and reprobed with an anti-HA antibody (bottom). (C) U2-OS cells were transfected with pHA-DYRK1Awt, pHA-DYRK1A/S520A, pHA-DYRK1A/K179R, or pHA-DYRK1A/Y310,312F, and soluble lysate fractions were incubated with unfused GST or GST-14-3-3β. Samples were analyzed by SDS-PAGE followed by immunoblotting with anti-HA antibody. Lysate represents 10% of input. (D) Quantification of the relative binding of different DYRK1A mutants to 14-3-3β compared with the fraction bound to DYRK1Awt is shown in the chart, which represents means ± SEM from three independent experiments. (E) A GFP-DYRK1A wild-type fusion protein (GFP-DYRK1Awt) and an HA-tagged DYRK1A kinase-inactive mutant (HA-DYRK1A/K179R) were individually expressed or coexpressed in U2-OS cells and detected by immunoblotting with anti-GFP and anti-HA antibodies, respectively (left). Phosphorylation of Ser-520 was analyzed by immunoblotting with the anti-DYRK1A pS520 antibody (middle). The expression levels of the two DYRK1A fusion proteins were compared by immunoblotting with anti-DYRK1A antibody (right).

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-08-0668

    Figure Lengend Snippet: DYRK1A autophosphorylates Ser-520 through an intramolecular reaction. (A) Bacterially expressed GST fusion proteins of wild-type DYRK1A (wt, lane 1) and the mutants DYRK1A/S520A (lane 2) and DYRK1A/K179R (lane 3) were immunoblotted with anti-DYRK1A pS520 (top), anti-phospho-Tyr (PY20; middle), and anti-GST (bottom) antibodies. The positions of marker proteins (in kilodaltons) are indicated. Arrows indicate full-length GST-fusion proteins, and asterisks indicate truncated GST-fusion proteins lacking the C-terminal (∼150) amino acids). The different patterns in DYRK1Awt, DYRK1AS520A, and DYRK1AK179R detected with the anti-GST antibody are due to differences in phosphorylation levels as shown in Supplemental Figure S3, A. (B) HA-tagged proteins corresponding to the two major isoforms of DYRK1A (lanes 1 and 2) and to distinct catalytically inactive point mutants (lanes 3–5) were overexpressed by transient transfection into U2-OS cells. Phosphorylation of Ser-520 was detected by immunoblotting with the phospho-specific anti-DYRK1A pS520 antibody (top). To control for equal expression of the fusion proteins, membranes were stripped and reprobed with an anti-HA antibody (bottom). (C) U2-OS cells were transfected with pHA-DYRK1Awt, pHA-DYRK1A/S520A, pHA-DYRK1A/K179R, or pHA-DYRK1A/Y310,312F, and soluble lysate fractions were incubated with unfused GST or GST-14-3-3β. Samples were analyzed by SDS-PAGE followed by immunoblotting with anti-HA antibody. Lysate represents 10% of input. (D) Quantification of the relative binding of different DYRK1A mutants to 14-3-3β compared with the fraction bound to DYRK1Awt is shown in the chart, which represents means ± SEM from three independent experiments. (E) A GFP-DYRK1A wild-type fusion protein (GFP-DYRK1Awt) and an HA-tagged DYRK1A kinase-inactive mutant (HA-DYRK1A/K179R) were individually expressed or coexpressed in U2-OS cells and detected by immunoblotting with anti-GFP and anti-HA antibodies, respectively (left). Phosphorylation of Ser-520 was analyzed by immunoblotting with the anti-DYRK1A pS520 antibody (middle). The expression levels of the two DYRK1A fusion proteins were compared by immunoblotting with anti-DYRK1A antibody (right).

    Article Snippet: Bacterial lysates were incubated with glutathione-Sepharose 4B beads (GE Healthcare) for 45 min at RT and washed four times with lysis buffer B. GST-DYRK1A fusion proteins were eluted with 10 mM reduced glutathione (Sigma-Aldrich) in 50 mM Tris-HCl, pH 8, and dialyzed against a buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM EDTA.

    Techniques: Marker, Transfection, Expressing, Incubation, SDS Page, Binding Assay, Mutagenesis

    Kinase activity of DYRK1A is modulated by the phosphorylation state of Ser-520. (A) US-OS cells were transfected with pHA-DYRK1Awt and the mutant pHA-DYRK1A/S520A. To determine the kinase activity of DYRK1A, tagged proteins were isolated by immunoprecipitation with anti-HA antibody, and immunocomplexes were subjected to in vitro kinase assay with DYRKtide as substrate. Equal amounts of HA-tagged proteins were used in all cases. The data represent means ± SEM from five independent experiments. **p ≤ 0.001. (B) U2-OS cells expressing HA-DYRK1Awt were treated during 8 h with the inhibitor SB216763 (50 μM) or with 0.1% DMSO as vehicle-control. Whole-cell extracts were resolved by 8% SDS-PAGE and immunoblotted with the anti-DYRK1A pS520 antibody (top). The membrane was then stripped and reprobed with anti-HA antibody to detect total HA-DYRK1A protein levels (bottom). (C) U2-OS cells expressing HA-DYRK1Awt were treated with increasing doses of SB216763, and soluble extracts were incubated with recombinant GST-14-3-3β. The amounts of bound DYRK1A (top) and DYRK1A in lysates (10% of inputs) (bottom) in each condition were analyzed by immunoblotting with anti-HA antibody. (D) The chart shows the quantification of the results expressed as the ratio of bound protein to input for each inhibitor concentration. (E) Kinase activities were determined as described in A for HA-DYRK1Awt and HA-DYRK1A/S520A expressed in U2-OS cells treated with 50 μM SB216763 for 8 h. For nontreated cells DMSO was used as vehicle-control. The data represent means ± SEM from three independent experiments. *p ≤ 0.05; **p ≤ 0.001.

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-08-0668

    Figure Lengend Snippet: Kinase activity of DYRK1A is modulated by the phosphorylation state of Ser-520. (A) US-OS cells were transfected with pHA-DYRK1Awt and the mutant pHA-DYRK1A/S520A. To determine the kinase activity of DYRK1A, tagged proteins were isolated by immunoprecipitation with anti-HA antibody, and immunocomplexes were subjected to in vitro kinase assay with DYRKtide as substrate. Equal amounts of HA-tagged proteins were used in all cases. The data represent means ± SEM from five independent experiments. **p ≤ 0.001. (B) U2-OS cells expressing HA-DYRK1Awt were treated during 8 h with the inhibitor SB216763 (50 μM) or with 0.1% DMSO as vehicle-control. Whole-cell extracts were resolved by 8% SDS-PAGE and immunoblotted with the anti-DYRK1A pS520 antibody (top). The membrane was then stripped and reprobed with anti-HA antibody to detect total HA-DYRK1A protein levels (bottom). (C) U2-OS cells expressing HA-DYRK1Awt were treated with increasing doses of SB216763, and soluble extracts were incubated with recombinant GST-14-3-3β. The amounts of bound DYRK1A (top) and DYRK1A in lysates (10% of inputs) (bottom) in each condition were analyzed by immunoblotting with anti-HA antibody. (D) The chart shows the quantification of the results expressed as the ratio of bound protein to input for each inhibitor concentration. (E) Kinase activities were determined as described in A for HA-DYRK1Awt and HA-DYRK1A/S520A expressed in U2-OS cells treated with 50 μM SB216763 for 8 h. For nontreated cells DMSO was used as vehicle-control. The data represent means ± SEM from three independent experiments. *p ≤ 0.05; **p ≤ 0.001.

    Article Snippet: Bacterial lysates were incubated with glutathione-Sepharose 4B beads (GE Healthcare) for 45 min at RT and washed four times with lysis buffer B. GST-DYRK1A fusion proteins were eluted with 10 mM reduced glutathione (Sigma-Aldrich) in 50 mM Tris-HCl, pH 8, and dialyzed against a buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM EDTA.

    Techniques: Activity Assay, Transfection, Mutagenesis, Isolation, Immunoprecipitation, In Vitro, Kinase Assay, Expressing, SDS Page, Incubation, Recombinant, Concentration Assay

    DYRK1A is phosphorylated on Ser-520 in vivo. (A) Whole-cell extracts from U2-OS cells expressing HA-DYRK1Awt were resolved by 8% SDS-PAGE and analyzed by immunoblot with anti-HA antibody (left) and with anti-pS520 antibody (right). Where indicated, the phosphorylated immunizing peptide (pS520: SNSGRARpSDPTHQHR) or an equivalent unphosphorylated peptide (S520: SNSGRARSDPTHQHR) was added at 25 μg/ml during the incubation period with 1 μg/ml anti-pS520 antibody. (B) Lysates from U2-OS cells transiently transfected with pHA-DYRK1A were incubated for 30 min at 30°C in phosphatase buffer alone (lanes 1 and 3) or supplemented with alkaline phosphatase (lanes 2 and 4), as indicated. Samples were analyzed by SDS-PAGE and immunoblot with anti-HA antibody (lanes 1 and 2) and with the anti-DYRK1A pS520 antibody (lanes 3 and 4). (C) U2-OS cells were transiently transfected with pHA-DYRK1Awt (lanes 1 and 3) and pHA-DYRK1A/S520A (lanes 2 and 4). Whole-cell extracts were resolved by 8% SDS-PAGE and immunoblotted with an anti-HA antibody (lanes 1 and 2) and with the anti-DYRK1A pS520 antibody (lanes 3 and 4). (D) Soluble cell extracts from U2-OS cells expressing HA-DYRK1Awt were incubated with GST-14-3-3β. Lysates, representing 10% of the input, (lane 1) and pulled-down proteins (lane 2) were separated by 8% SDS-PAGE and analyzed by immunoblot with anti-HA antibody (bottom) and with the phospho-specific anti-DYRK1A pS520 antibody (top) to detect the presence of Ser-520–phosphorylated DYRK1A bound to 14-3-3β. (E) The endogenous DYRK1A protein was immunoprecipitated from PC12 cells with anti-DYRK1A antibody. The immunoprecipitate was analyzed by immunoblot (8% SDS-PAGE) with both the anti-DYRK1A and the anti-DYRK1A pS520 antibody. The position of marker proteins (in kilodaltons) is indicated.

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-08-0668

    Figure Lengend Snippet: DYRK1A is phosphorylated on Ser-520 in vivo. (A) Whole-cell extracts from U2-OS cells expressing HA-DYRK1Awt were resolved by 8% SDS-PAGE and analyzed by immunoblot with anti-HA antibody (left) and with anti-pS520 antibody (right). Where indicated, the phosphorylated immunizing peptide (pS520: SNSGRARpSDPTHQHR) or an equivalent unphosphorylated peptide (S520: SNSGRARSDPTHQHR) was added at 25 μg/ml during the incubation period with 1 μg/ml anti-pS520 antibody. (B) Lysates from U2-OS cells transiently transfected with pHA-DYRK1A were incubated for 30 min at 30°C in phosphatase buffer alone (lanes 1 and 3) or supplemented with alkaline phosphatase (lanes 2 and 4), as indicated. Samples were analyzed by SDS-PAGE and immunoblot with anti-HA antibody (lanes 1 and 2) and with the anti-DYRK1A pS520 antibody (lanes 3 and 4). (C) U2-OS cells were transiently transfected with pHA-DYRK1Awt (lanes 1 and 3) and pHA-DYRK1A/S520A (lanes 2 and 4). Whole-cell extracts were resolved by 8% SDS-PAGE and immunoblotted with an anti-HA antibody (lanes 1 and 2) and with the anti-DYRK1A pS520 antibody (lanes 3 and 4). (D) Soluble cell extracts from U2-OS cells expressing HA-DYRK1Awt were incubated with GST-14-3-3β. Lysates, representing 10% of the input, (lane 1) and pulled-down proteins (lane 2) were separated by 8% SDS-PAGE and analyzed by immunoblot with anti-HA antibody (bottom) and with the phospho-specific anti-DYRK1A pS520 antibody (top) to detect the presence of Ser-520–phosphorylated DYRK1A bound to 14-3-3β. (E) The endogenous DYRK1A protein was immunoprecipitated from PC12 cells with anti-DYRK1A antibody. The immunoprecipitate was analyzed by immunoblot (8% SDS-PAGE) with both the anti-DYRK1A and the anti-DYRK1A pS520 antibody. The position of marker proteins (in kilodaltons) is indicated.

    Article Snippet: Bacterial lysates were incubated with glutathione-Sepharose 4B beads (GE Healthcare) for 45 min at RT and washed four times with lysis buffer B. GST-DYRK1A fusion proteins were eluted with 10 mM reduced glutathione (Sigma-Aldrich) in 50 mM Tris-HCl, pH 8, and dialyzed against a buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM EDTA.

    Techniques: In Vivo, Expressing, SDS Page, Incubation, Transfection, Immunoprecipitation, Marker

    14-3-3β binding to phospho-Ser-520 regulates DYRK1A kinase activity in vitro. (A) Kinase activity of recombinant DYRK1A proteins (GST-DYRK1Awt and the mutants S520A and K179R) was assayed with DYRKtide as substrate. The kinetics of the reaction indicated that 32 P incorporation was linear with time and with the enzyme amount used in the assay. (B) The activity of GST-DYRK1Awt versus GST-DYRK1A/S520A is represented as the means ± SEM from three independent experiments, with each point measured in triplicate in the individual experiments. (C) Kinase activities of the indicated GST-DYRK1A proteins were assayed as described in A with increasing amounts of recombinant GST-14-3-3β (0.1, 0.5, and 1 μg) or GST alone (1 μg). The assay was performed three times, giving similar results in each; a representative assay is shown, and the data correspond to the means ± SEM of triplicate measurements.

    Journal: Molecular Biology of the Cell

    Article Title:

    doi: 10.1091/mbc.E06-08-0668

    Figure Lengend Snippet: 14-3-3β binding to phospho-Ser-520 regulates DYRK1A kinase activity in vitro. (A) Kinase activity of recombinant DYRK1A proteins (GST-DYRK1Awt and the mutants S520A and K179R) was assayed with DYRKtide as substrate. The kinetics of the reaction indicated that 32 P incorporation was linear with time and with the enzyme amount used in the assay. (B) The activity of GST-DYRK1Awt versus GST-DYRK1A/S520A is represented as the means ± SEM from three independent experiments, with each point measured in triplicate in the individual experiments. (C) Kinase activities of the indicated GST-DYRK1A proteins were assayed as described in A with increasing amounts of recombinant GST-14-3-3β (0.1, 0.5, and 1 μg) or GST alone (1 μg). The assay was performed three times, giving similar results in each; a representative assay is shown, and the data correspond to the means ± SEM of triplicate measurements.

    Article Snippet: Bacterial lysates were incubated with glutathione-Sepharose 4B beads (GE Healthcare) for 45 min at RT and washed four times with lysis buffer B. GST-DYRK1A fusion proteins were eluted with 10 mM reduced glutathione (Sigma-Aldrich) in 50 mM Tris-HCl, pH 8, and dialyzed against a buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM EDTA.

    Techniques: Binding Assay, Activity Assay, In Vitro, Recombinant