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Roche nonidet p40 buffer
Nonidet P40 Buffer, 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/nonidet p40 buffer/product/Roche
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nonidet p40 buffer - by Bioz Stars, 2020-04
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Related Articles

In Vivo:

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. For in vitro and in vivo binding, immobilized GST fusion proteins (made in BL21/DE3 cells) were incubated with bacterially purified Flag-TIP49A and Flag-TIP49B or with protein expressed in mammalian cells.

In Vitro:

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. For in vitro and in vivo binding, immobilized GST fusion proteins (made in BL21/DE3 cells) were incubated with bacterially purified Flag-TIP49A and Flag-TIP49B or with protein expressed in mammalian cells.

Protease Inhibitor:

Article Title: PTK2-mediated degradation of ATG3 impedes cancer cells susceptible to DNA damage treatment
Article Snippet: .. For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer (1% Nonidet P40 [Amersco, E109], 150 mM NaCl, 50 mM Tris at pH 7.5, and 5 mM EDTA) supplemented with complete protease inhibitor cocktail (Roche, 5892791001) and phosphatase inhibitor cocktail (Applygen, P1260). .. Whole-cell lysate proteins were used for immunoprecipitation with the indicated antibodies.

Article Title: FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway
Article Snippet: .. For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer supplemented with a complete protease inhibitor cocktail (Roche, 04693132001). .. Whole-cell lysates or cytosolic proteins were used for immunoprecipitation with the indicated antibodies.

Article Title: Targeted polyubiquitylation of RASSF1C by the Mule and SCFβ-TrCP ligases in response to DNA damage
Article Snippet: .. Ten 15 cm plates of HEK-293T SBP-FLAG-RASSF1C stable cells were pretreated with MG132 for 6 h before harvest, and then lysed in 40 ml of 0.5% Nonidet P40 buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl and 0.5% Nonidet P40) containing 10 mM NaF, 1 mM Na3 VO4 and protease inhibitor mixture (Roche) for 30 min. SBP-FLAG-RASSF1C in the supernatant was precipitated for 3 h with 100 µl streptavidin resin (GE HealthCare, 17–5113-01), which was then washed three times with 0.5% Nonidet P40 buffer followed by three washes with 50 mM NH4 HCO3 . ..

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. Beads were washed with high-salt washing buffer (50 mM Tris (pH 7.3), 500 mM NaCl, 0.1% (v/v) Nonidet P-40, 200 µg ml−1 BSA, 0.5 mM DTT, 1 mM PMSF and Roche protease inhibitor cocktail), then subjected to SDS-PAGE and western blotting.

Centrifugation:

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: .. Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. For in vitro and in vivo binding, immobilized GST fusion proteins (made in BL21/DE3 cells) were incubated with bacterially purified Flag-TIP49A and Flag-TIP49B or with protein expressed in mammalian cells.

Purification:

Article Title: Targeted polyubiquitylation of RASSF1C by the Mule and SCFβ-TrCP ligases in response to DNA damage
Article Snippet: Paragraph title: SBP purification of RASSF1C protein complexes ... Ten 15 cm plates of HEK-293T SBP-FLAG-RASSF1C stable cells were pretreated with MG132 for 6 h before harvest, and then lysed in 40 ml of 0.5% Nonidet P40 buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl and 0.5% Nonidet P40) containing 10 mM NaF, 1 mM Na3 VO4 and protease inhibitor mixture (Roche) for 30 min. SBP-FLAG-RASSF1C in the supernatant was precipitated for 3 h with 100 µl streptavidin resin (GE HealthCare, 17–5113-01), which was then washed three times with 0.5% Nonidet P40 buffer followed by three washes with 50 mM NH4 HCO3 .

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. For in vitro and in vivo binding, immobilized GST fusion proteins (made in BL21/DE3 cells) were incubated with bacterially purified Flag-TIP49A and Flag-TIP49B or with protein expressed in mammalian cells.

Immunoprecipitation:

Article Title: PTK2-mediated degradation of ATG3 impedes cancer cells susceptible to DNA damage treatment
Article Snippet: .. For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer (1% Nonidet P40 [Amersco, E109], 150 mM NaCl, 50 mM Tris at pH 7.5, and 5 mM EDTA) supplemented with complete protease inhibitor cocktail (Roche, 5892791001) and phosphatase inhibitor cocktail (Applygen, P1260). .. Whole-cell lysate proteins were used for immunoprecipitation with the indicated antibodies.

Article Title: FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway
Article Snippet: .. For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer supplemented with a complete protease inhibitor cocktail (Roche, 04693132001). .. Whole-cell lysates or cytosolic proteins were used for immunoprecipitation with the indicated antibodies.

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: .. Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. For in vitro and in vivo binding, immobilized GST fusion proteins (made in BL21/DE3 cells) were incubated with bacterially purified Flag-TIP49A and Flag-TIP49B or with protein expressed in mammalian cells.

Incubation:

Article Title: PTK2-mediated degradation of ATG3 impedes cancer cells susceptible to DNA damage treatment
Article Snippet: For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer (1% Nonidet P40 [Amersco, E109], 150 mM NaCl, 50 mM Tris at pH 7.5, and 5 mM EDTA) supplemented with complete protease inhibitor cocktail (Roche, 5892791001) and phosphatase inhibitor cocktail (Applygen, P1260). .. Generally, 2 μg of antibody was added to 1 ml of cell lysate, which was incubated at 4°C for 8 to 12 h. After the addition of protein G-agarose beads (GE Healthcare, 17–0618–01), the incubation was continued for 2 h. Immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min. All bands of blots were scanned with a phosphorimager, and the relative intensity of each band was normalized to each band of ACTB.

Article Title: FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway
Article Snippet: For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer supplemented with a complete protease inhibitor cocktail (Roche, 04693132001). .. Generally, 1 to 4 µg of antibody was added to 1 ml of cell lysate, which was incubated at 4°C for 8 to12 h. After the addition of Protein A/G-agarose beads, the incubation was continued for 1 h. Immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min. Main western blotting data showing MAP1LC3-II accumulation or SQSTM1 turnover.

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. For in vitro and in vivo binding, immobilized GST fusion proteins (made in BL21/DE3 cells) were incubated with bacterially purified Flag-TIP49A and Flag-TIP49B or with protein expressed in mammalian cells.

Transfection:

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Protein interactions were examined in 293T cells 36 h after Lipofectamine 2000–mediated transfection of the indicated plasmids. .. Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation.

Lysis:

Article Title: PTK2-mediated degradation of ATG3 impedes cancer cells susceptible to DNA damage treatment
Article Snippet: For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer (1% Nonidet P40 [Amersco, E109], 150 mM NaCl, 50 mM Tris at pH 7.5, and 5 mM EDTA) supplemented with complete protease inhibitor cocktail (Roche, 5892791001) and phosphatase inhibitor cocktail (Applygen, P1260). .. Generally, 2 μg of antibody was added to 1 ml of cell lysate, which was incubated at 4°C for 8 to 12 h. After the addition of protein G-agarose beads (GE Healthcare, 17–0618–01), the incubation was continued for 2 h. Immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min. All bands of blots were scanned with a phosphorimager, and the relative intensity of each band was normalized to each band of ACTB.

Article Title: FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway
Article Snippet: For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer supplemented with a complete protease inhibitor cocktail (Roche, 04693132001). .. Generally, 1 to 4 µg of antibody was added to 1 ml of cell lysate, which was incubated at 4°C for 8 to12 h. After the addition of Protein A/G-agarose beads, the incubation was continued for 1 h. Immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min. Main western blotting data showing MAP1LC3-II accumulation or SQSTM1 turnover.

Western Blot:

Article Title: FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway
Article Snippet: For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer supplemented with a complete protease inhibitor cocktail (Roche, 04693132001). .. Generally, 1 to 4 µg of antibody was added to 1 ml of cell lysate, which was incubated at 4°C for 8 to12 h. After the addition of Protein A/G-agarose beads, the incubation was continued for 1 h. Immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min. Main western blotting data showing MAP1LC3-II accumulation or SQSTM1 turnover.

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. Beads were washed with high-salt washing buffer (50 mM Tris (pH 7.3), 500 mM NaCl, 0.1% (v/v) Nonidet P-40, 200 µg ml−1 BSA, 0.5 mM DTT, 1 mM PMSF and Roche protease inhibitor cocktail), then subjected to SDS-PAGE and western blotting.

Binding Assay:

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. For in vitro and in vivo binding, immobilized GST fusion proteins (made in BL21/DE3 cells) were incubated with bacterially purified Flag-TIP49A and Flag-TIP49B or with protein expressed in mammalian cells.

SDS Page:

Article Title: PTK2-mediated degradation of ATG3 impedes cancer cells susceptible to DNA damage treatment
Article Snippet: Equal amounts of proteins were size-fractionated by 7.5–15% SDS-PAGE. .. For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer (1% Nonidet P40 [Amersco, E109], 150 mM NaCl, 50 mM Tris at pH 7.5, and 5 mM EDTA) supplemented with complete protease inhibitor cocktail (Roche, 5892791001) and phosphatase inhibitor cocktail (Applygen, P1260).

Article Title: FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway
Article Snippet: Equal amounts of proteins (100 to150 µg) were size-fractionated by 7.5–15% SDS-PAGE. .. For immunoprecipitation, cells were harvested and then lysed in a Nonidet P40 buffer supplemented with a complete protease inhibitor cocktail (Roche, 04693132001).

Article Title: A YY1-INO80 complex regulates genomic stability through homologous recombination-based repair
Article Snippet: Cells were lysed in 0.1% (v/v) Nonidet P40 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) and cleared by centrifugation (14,000 r.p.m. for 15 min) before immunoprecipitation. .. Beads were washed with high-salt washing buffer (50 mM Tris (pH 7.3), 500 mM NaCl, 0.1% (v/v) Nonidet P-40, 200 µg ml−1 BSA, 0.5 mM DTT, 1 mM PMSF and Roche protease inhibitor cocktail), then subjected to SDS-PAGE and western blotting.

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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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    88
    Roche nt rna immunoprecipitation buffer
    Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by <t>RNA</t> <t>immunoprecipitation</t> assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008
    Nt Rna Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nt rna immunoprecipitation buffer/product/Roche
    Average 88 stars, based on 1 article reviews
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    nt rna immunoprecipitation buffer - by Bioz Stars, 2020-04
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    86
    Roche cell lysis ripa buffer
    Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by <t>RNA</t> <t>immunoprecipitation</t> assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008
    Cell Lysis Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell lysis ripa buffer/product/Roche
    Average 86 stars, based on 1 article reviews
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    99
    Roche lysis buffer
    Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by <t>RNA</t> <t>immunoprecipitation</t> assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008
    Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 1428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lysis buffer/product/Roche
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    Image Search Results


    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

    Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008

    Journal: eLife

    Article Title: RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response

    doi: 10.7554/eLife.09656

    Figure Lengend Snippet: Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008

    Article Snippet: After homogenization in 100 μl of NT- RNA immunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.5% Nonidet P40, 1 mM EDTA pH 8.0, 1 mM DTT), Complete protease inhibitor (Roche), 200 U/ml RNase Out (Invitrogen, USA) samples were incubated for 10 min on ice and pre-cleared with 25 μl of Protein G-coated Dynabeads (Life Technologies).

    Techniques: Binding Assay, Immunoprecipitation, Incubation, Quantitative RT-PCR