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Roche sodium deoxycholate protease inhibitors
Sodium Deoxycholate Protease Inhibitors, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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sodium deoxycholate protease inhibitors - by Bioz Stars, 2020-04
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Transfection:

Article Title: Simian-Human Immunodeficiency Virus Escape from Cytotoxic T-Lymphocyte Recognition at a Structurally Constrained Epitope
Article Snippet: 293T cells were transfected with 1 μg of purified gag expression plasmid by using a CellPhect transfection kit (Amersham Pharmacia, Piscataway, N.J.). .. Forty-eight hours later, cell supernatants were collected, and cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P-40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

Article Title: Compensatory Substitutions Restore Normal Core Assembly in Simian Immunodeficiency Virus Isolates with Gag Epitope Cytotoxic T-Lymphocyte Escape Mutations
Article Snippet: 293T cells were transfected with 1 μg of purified gag expression plasmid by the calcium phosphate method (Invitrogen, Carlsbad, Calif.). .. Forty-eight hours later, cell supernatants were collected and the cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

Protease Inhibitor:

Article Title: An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
Article Snippet: .. Laemmli buffer (4× sample buffer) 0.2 M Tris-HCl pH 6.8 40% glycerol 277 mM SDS 200 mM DTT 3 mM bromophenol blue Add all components to ddH2 O; aliquot into 1.5 ml microcentrifuge tubes and store at −20 °C Coomassie stain 1.2 g Coomassie blue 300 ml methanol 60 ml acetic acid 240 ml H2 O Add all components; store at room temperature TBS 50 mM Tris-HCl pH 7.5 300 mM NaCl Add all components to ddH2 O and bring to the desired volume (usually make 20 L at a time in a carboy) Store at room temperature TBST 50 mM Tris-HCl pH 7.5 300 mM NaCl 0.05% Tween 20 Add all components to ddH2 O and bring to the desired volume (usually make 20 L at a time in a carboy) Store at room temperature 140 mM ChIP lysis buffer 50 mM HEPES pH 7.5 140 mM NaCl 1% Triton X-100 0.1% sodium deoxycholate Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride* 2.0 mM benzamidine* 2.0 μM pepstatin* 0.6 μM leupeptin* 2.0 μg of chymostatin* *Note: Per ml of buffer . ..

Purification:

Article Title: Simian-Human Immunodeficiency Virus Escape from Cytotoxic T-Lymphocyte Recognition at a Structurally Constrained Epitope
Article Snippet: 293T cells were transfected with 1 μg of purified gag expression plasmid by using a CellPhect transfection kit (Amersham Pharmacia, Piscataway, N.J.). .. Forty-eight hours later, cell supernatants were collected, and cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P-40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

Article Title: Compensatory Substitutions Restore Normal Core Assembly in Simian Immunodeficiency Virus Isolates with Gag Epitope Cytotoxic T-Lymphocyte Escape Mutations
Article Snippet: 293T cells were transfected with 1 μg of purified gag expression plasmid by the calcium phosphate method (Invitrogen, Carlsbad, Calif.). .. Forty-eight hours later, cell supernatants were collected and the cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

Enzyme-linked Immunosorbent Assay:

Article Title: Compensatory Substitutions Restore Normal Core Assembly in Simian Immunodeficiency Virus Isolates with Gag Epitope Cytotoxic T-Lymphocyte Escape Mutations
Article Snippet: Forty-eight hours later, cell supernatants were collected and the cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany). .. Concentrations of SIV p27 in the supernatant were quantified using the SIV core antigen enzyme-linked immunosorbent assay (ELISA) (Coulter, Miami, Fla.).

Expressing:

Article Title: Simian-Human Immunodeficiency Virus Escape from Cytotoxic T-Lymphocyte Recognition at a Structurally Constrained Epitope
Article Snippet: 293T cells were transfected with 1 μg of purified gag expression plasmid by using a CellPhect transfection kit (Amersham Pharmacia, Piscataway, N.J.). .. Forty-eight hours later, cell supernatants were collected, and cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P-40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

Article Title: Compensatory Substitutions Restore Normal Core Assembly in Simian Immunodeficiency Virus Isolates with Gag Epitope Cytotoxic T-Lymphocyte Escape Mutations
Article Snippet: 293T cells were transfected with 1 μg of purified gag expression plasmid by the calcium phosphate method (Invitrogen, Carlsbad, Calif.). .. Forty-eight hours later, cell supernatants were collected and the cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

Staining:

Article Title: An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
Article Snippet: .. Laemmli buffer (4× sample buffer) 0.2 M Tris-HCl pH 6.8 40% glycerol 277 mM SDS 200 mM DTT 3 mM bromophenol blue Add all components to ddH2 O; aliquot into 1.5 ml microcentrifuge tubes and store at −20 °C Coomassie stain 1.2 g Coomassie blue 300 ml methanol 60 ml acetic acid 240 ml H2 O Add all components; store at room temperature TBS 50 mM Tris-HCl pH 7.5 300 mM NaCl Add all components to ddH2 O and bring to the desired volume (usually make 20 L at a time in a carboy) Store at room temperature TBST 50 mM Tris-HCl pH 7.5 300 mM NaCl 0.05% Tween 20 Add all components to ddH2 O and bring to the desired volume (usually make 20 L at a time in a carboy) Store at room temperature 140 mM ChIP lysis buffer 50 mM HEPES pH 7.5 140 mM NaCl 1% Triton X-100 0.1% sodium deoxycholate Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride* 2.0 mM benzamidine* 2.0 μM pepstatin* 0.6 μM leupeptin* 2.0 μg of chymostatin* *Note: Per ml of buffer . ..

Western Blot:

Article Title: Simian-Human Immunodeficiency Virus Escape from Cytotoxic T-Lymphocyte Recognition at a Structurally Constrained Epitope
Article Snippet: Paragraph title: Protein quantification and Western blotting. ... Forty-eight hours later, cell supernatants were collected, and cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P-40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

Lysis:

Article Title: An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
Article Snippet: .. Laemmli buffer (4× sample buffer) 0.2 M Tris-HCl pH 6.8 40% glycerol 277 mM SDS 200 mM DTT 3 mM bromophenol blue Add all components to ddH2 O; aliquot into 1.5 ml microcentrifuge tubes and store at −20 °C Coomassie stain 1.2 g Coomassie blue 300 ml methanol 60 ml acetic acid 240 ml H2 O Add all components; store at room temperature TBS 50 mM Tris-HCl pH 7.5 300 mM NaCl Add all components to ddH2 O and bring to the desired volume (usually make 20 L at a time in a carboy) Store at room temperature TBST 50 mM Tris-HCl pH 7.5 300 mM NaCl 0.05% Tween 20 Add all components to ddH2 O and bring to the desired volume (usually make 20 L at a time in a carboy) Store at room temperature 140 mM ChIP lysis buffer 50 mM HEPES pH 7.5 140 mM NaCl 1% Triton X-100 0.1% sodium deoxycholate Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride* 2.0 mM benzamidine* 2.0 μM pepstatin* 0.6 μM leupeptin* 2.0 μg of chymostatin* *Note: Per ml of buffer . ..

Chromatin Immunoprecipitation:

Article Title: An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
Article Snippet: .. Laemmli buffer (4× sample buffer) 0.2 M Tris-HCl pH 6.8 40% glycerol 277 mM SDS 200 mM DTT 3 mM bromophenol blue Add all components to ddH2 O; aliquot into 1.5 ml microcentrifuge tubes and store at −20 °C Coomassie stain 1.2 g Coomassie blue 300 ml methanol 60 ml acetic acid 240 ml H2 O Add all components; store at room temperature TBS 50 mM Tris-HCl pH 7.5 300 mM NaCl Add all components to ddH2 O and bring to the desired volume (usually make 20 L at a time in a carboy) Store at room temperature TBST 50 mM Tris-HCl pH 7.5 300 mM NaCl 0.05% Tween 20 Add all components to ddH2 O and bring to the desired volume (usually make 20 L at a time in a carboy) Store at room temperature 140 mM ChIP lysis buffer 50 mM HEPES pH 7.5 140 mM NaCl 1% Triton X-100 0.1% sodium deoxycholate Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride* 2.0 mM benzamidine* 2.0 μM pepstatin* 0.6 μM leupeptin* 2.0 μg of chymostatin* *Note: Per ml of buffer . ..

Plasmid Preparation:

Article Title: Simian-Human Immunodeficiency Virus Escape from Cytotoxic T-Lymphocyte Recognition at a Structurally Constrained Epitope
Article Snippet: 293T cells were transfected with 1 μg of purified gag expression plasmid by using a CellPhect transfection kit (Amersham Pharmacia, Piscataway, N.J.). .. Forty-eight hours later, cell supernatants were collected, and cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P-40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

Article Title: Compensatory Substitutions Restore Normal Core Assembly in Simian Immunodeficiency Virus Isolates with Gag Epitope Cytotoxic T-Lymphocyte Escape Mutations
Article Snippet: 293T cells were transfected with 1 μg of purified gag expression plasmid by the calcium phosphate method (Invitrogen, Carlsbad, Calif.). .. Forty-eight hours later, cell supernatants were collected and the cells were lysed for 20 min with 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-1% Nonidet P40-0.5% sodium deoxycholate-protease inhibitors (Roche, Mannheim, Germany).

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  • 99
    Roche radio immunoprecipitation assay buffer
    <t>Immunoprecipitation</t> with anti-P2X 1 antibody. Antibodies to P2X 1 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads were resolved by SDS-PAGE, and Western blots were probed with A) P2X 1, B) P2X 4 or C) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. Monomeric P2X 1 can be seen highly concentrated in the pulldown fraction at 50 kDa. Little P2X 1 appears in FT. ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type, but is completely absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to possibly P2X 1 . Note, there is no evidence of the 70 kDa band in the IP lane. ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 1 is ‘clean’ with no non-specific protein binding evident in the IP lanes.
    Radio Immunoprecipitation Assay Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/radio immunoprecipitation assay buffer/product/Roche
    Average 99 stars, based on 69 article reviews
    Price from $9.99 to $1999.99
    radio immunoprecipitation assay buffer - by Bioz Stars, 2020-04
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    99
    Roche ripa buffer
    Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in <t>293T</t> cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with <t>RIPA</t> buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.
    Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 4719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ripa buffer/product/Roche
    Average 99 stars, based on 4719 article reviews
    Price from $9.99 to $1999.99
    ripa buffer - by Bioz Stars, 2020-04
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    91
    Roche deoxycholate doc insoluble fibronectin
    ECM formed through LPA 1 signaling supports the proliferation of chondrocytes. ( a,b ) FN deposition is enhanced by LPA 1 signaling. ( a ) Chondrocytes were cultured in medium containing 10% FCS for 10 days and immunostained with anti-FN antibody. Scale bar: 10 μm. ( b ) <t>Deoxycholate-insoluble</t> <t>fibronectin</t> in the extracellular matrix (ECM) was detected by western blot. ( c ) Comparison of ECM amount between HT and KO chondrocytes. ( d ) FN and Col II are similarly expressed in HT and KO chondrocytes. (Data are mean ± s.d., n = 3, N.S.: not significant) ( e ) ECM formed through LPA 1 signaling supports the cell proliferation efficiently. HT chondrocytes were cultured on the decellularized-ECM plates which formed either by HT chondrocytes (ECM formed by HT) or KO chondrocytes (ECM formed by KO), and time-dependent cell proliferations were determined. (Data are mean ± s.d., n = 3, *** P
    Deoxycholate Doc Insoluble Fibronectin, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxycholate doc insoluble fibronectin/product/Roche
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Immunoprecipitation with anti-P2X 1 antibody. Antibodies to P2X 1 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads were resolved by SDS-PAGE, and Western blots were probed with A) P2X 1, B) P2X 4 or C) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. Monomeric P2X 1 can be seen highly concentrated in the pulldown fraction at 50 kDa. Little P2X 1 appears in FT. ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type, but is completely absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to possibly P2X 1 . Note, there is no evidence of the 70 kDa band in the IP lane. ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 1 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

    Journal: Scientific Reports

    Article Title: Role of P2X4 Receptor in Mouse Voiding Function

    doi: 10.1038/s41598-018-20216-4

    Figure Lengend Snippet: Immunoprecipitation with anti-P2X 1 antibody. Antibodies to P2X 1 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads were resolved by SDS-PAGE, and Western blots were probed with A) P2X 1, B) P2X 4 or C) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. Monomeric P2X 1 can be seen highly concentrated in the pulldown fraction at 50 kDa. Little P2X 1 appears in FT. ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type, but is completely absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to possibly P2X 1 . Note, there is no evidence of the 70 kDa band in the IP lane. ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 1 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

    Article Snippet: Western Blot Excised whole bladders were put in 0.5 ml ice-cold radio immunoprecipitation assay buffer (RIPA; 50 mM Tris pH 8.0, 150 mM NaCl, 1% v/v NP-40, 0.5% w/v deoxycholic acid, 0.1% w/v SDS) containing Complete Mini Protease Inhibitor Cocktail tablets (Roche, Germany).

    Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Mouse Assay, Protein Binding

    Immunoprecipitation with anti-P2X 4 antibody. Antibodies to P2X 4 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads, were resolved by SDS-PAGE, and Western blots were probed with A ) P2X 1, B) P2X 4 or C ) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. P2X 1 is highly concentrated in the FT fractions. Minor potential P2X 1 staining appears in the IP lane, however this is due to P2X 4 antibody cross-reacting and pulling down some P2X 1 . ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type IP lane, but is absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to P2X 1 (50 kDa band). ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 4 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

    Journal: Scientific Reports

    Article Title: Role of P2X4 Receptor in Mouse Voiding Function

    doi: 10.1038/s41598-018-20216-4

    Figure Lengend Snippet: Immunoprecipitation with anti-P2X 4 antibody. Antibodies to P2X 4 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads, were resolved by SDS-PAGE, and Western blots were probed with A ) P2X 1, B) P2X 4 or C ) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. P2X 1 is highly concentrated in the FT fractions. Minor potential P2X 1 staining appears in the IP lane, however this is due to P2X 4 antibody cross-reacting and pulling down some P2X 1 . ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type IP lane, but is absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to P2X 1 (50 kDa band). ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 4 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

    Article Snippet: Western Blot Excised whole bladders were put in 0.5 ml ice-cold radio immunoprecipitation assay buffer (RIPA; 50 mM Tris pH 8.0, 150 mM NaCl, 1% v/v NP-40, 0.5% w/v deoxycholic acid, 0.1% w/v SDS) containing Complete Mini Protease Inhibitor Cocktail tablets (Roche, Germany).

    Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Mouse Assay, Staining, Protein Binding

    Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

    Journal: Oncotarget

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    doi: 10.18632/oncotarget.8072

    Figure Lengend Snippet: Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

    Article Snippet: After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany).

    Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Staining, Plasmid Preparation, Mutagenesis, Transfection

    Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

    Journal: Oncotarget

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    doi: 10.18632/oncotarget.8072

    Figure Lengend Snippet: Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

    Article Snippet: After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany).

    Techniques: Incubation, Immunoprecipitation, Transfection, Western Blot

    Expression of TM6SF2 in cultured hepatocytes. ( a ) Plasmids encoding wild-type and mutant human TM6SF2 were expressed in HuH7 cells. Two days after transfection, the TM6SF2 mRNA levels were measured using Real-Time PCR (left). The cells were harvested and solubilized in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH=8). Quantitative immunoblotting was performed using a LI-COR Odyssey infrared imaging system as described in the Methods (right). The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments. V, vector. ( b ) Recombinant wild-type hTM6SF2 was expressed in Hepa1c1c7 cells. After two days, the cells were fractionated and subjected to immunoblotting as described in the Methods. C, cytosol; M, membranes; LD, lipid droplets; L, whole cell lysate. The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments.

    Journal: Nature genetics

    Article Title: Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease

    doi: 10.1038/ng.2901

    Figure Lengend Snippet: Expression of TM6SF2 in cultured hepatocytes. ( a ) Plasmids encoding wild-type and mutant human TM6SF2 were expressed in HuH7 cells. Two days after transfection, the TM6SF2 mRNA levels were measured using Real-Time PCR (left). The cells were harvested and solubilized in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH=8). Quantitative immunoblotting was performed using a LI-COR Odyssey infrared imaging system as described in the Methods (right). The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments. V, vector. ( b ) Recombinant wild-type hTM6SF2 was expressed in Hepa1c1c7 cells. After two days, the cells were fractionated and subjected to immunoblotting as described in the Methods. C, cytosol; M, membranes; LD, lipid droplets; L, whole cell lysate. The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments.

    Article Snippet: The other aliquot was suspended in RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) supplemented with protease inhibitors (Protease Inhibitor Cocktail, Roche) and disrupted by 20 passages through a 25-gauge needle.

    Techniques: Expressing, Cell Culture, Mutagenesis, Transfection, Real-time Polymerase Chain Reaction, Imaging, Plasmid Preparation, Recombinant

    ECM formed through LPA 1 signaling supports the proliferation of chondrocytes. ( a,b ) FN deposition is enhanced by LPA 1 signaling. ( a ) Chondrocytes were cultured in medium containing 10% FCS for 10 days and immunostained with anti-FN antibody. Scale bar: 10 μm. ( b ) Deoxycholate-insoluble fibronectin in the extracellular matrix (ECM) was detected by western blot. ( c ) Comparison of ECM amount between HT and KO chondrocytes. ( d ) FN and Col II are similarly expressed in HT and KO chondrocytes. (Data are mean ± s.d., n = 3, N.S.: not significant) ( e ) ECM formed through LPA 1 signaling supports the cell proliferation efficiently. HT chondrocytes were cultured on the decellularized-ECM plates which formed either by HT chondrocytes (ECM formed by HT) or KO chondrocytes (ECM formed by KO), and time-dependent cell proliferations were determined. (Data are mean ± s.d., n = 3, *** P

    Journal: Scientific Reports

    Article Title: ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation

    doi: 10.1038/srep23433

    Figure Lengend Snippet: ECM formed through LPA 1 signaling supports the proliferation of chondrocytes. ( a,b ) FN deposition is enhanced by LPA 1 signaling. ( a ) Chondrocytes were cultured in medium containing 10% FCS for 10 days and immunostained with anti-FN antibody. Scale bar: 10 μm. ( b ) Deoxycholate-insoluble fibronectin in the extracellular matrix (ECM) was detected by western blot. ( c ) Comparison of ECM amount between HT and KO chondrocytes. ( d ) FN and Col II are similarly expressed in HT and KO chondrocytes. (Data are mean ± s.d., n = 3, N.S.: not significant) ( e ) ECM formed through LPA 1 signaling supports the cell proliferation efficiently. HT chondrocytes were cultured on the decellularized-ECM plates which formed either by HT chondrocytes (ECM formed by HT) or KO chondrocytes (ECM formed by KO), and time-dependent cell proliferations were determined. (Data are mean ± s.d., n = 3, *** P

    Article Snippet: Western blot analysis of deoxycholate (DOC)-insoluble fibronectin After 10 days culture, cells were washed with PBS and solubilized with deoxycholate lysis buffer (DOC-buffer) containing 2% sodium deoxycholate, protease inhibitors (Complete Protease Inhibitor Cocktail, Roche), 20 mM Tris-HCl pH 8.8, 2 mM EDTA, 2 mM iodoacetamide and 2 mM N-ethylmaleimide.

    Techniques: Cell Culture, Western Blot