ribonuclease inhibitor  (Roche)


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    Structured Review

    Roche ribonuclease inhibitor
    Ribonuclease Inhibitor, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ribonuclease inhibitor/product/Roche
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    Lysis:

    Article Title: Proteomics-Based Characterization of miR-574-5p Decoy to CUGBP1 Suggests Specificity for mPGES-1 Regulation in Human Lung Cancer Cells
    Article Snippet: .. In short, 6 × 106 A549 cells per RIP were resuspended in lysis buffer containing 10 mM Tris-HCl (Carl Roth) pH 7.5, 10 mM KCl (Sigma-Aldrich), 1.5 mM MgCl2, 0.5 mM DTT (Sigma-Aldrich), 0.9% Nonidet P-40 (Sigma-Aldrich), 20 μl ribonuclease inhibitor and protease inhibitor EDTA-free (Roche). .. We used blocked GammaBind Plus Sepharose beads (GE Healthcare).

    Protease Inhibitor:

    Article Title: Proteomics-Based Characterization of miR-574-5p Decoy to CUGBP1 Suggests Specificity for mPGES-1 Regulation in Human Lung Cancer Cells
    Article Snippet: .. In short, 6 × 106 A549 cells per RIP were resuspended in lysis buffer containing 10 mM Tris-HCl (Carl Roth) pH 7.5, 10 mM KCl (Sigma-Aldrich), 1.5 mM MgCl2, 0.5 mM DTT (Sigma-Aldrich), 0.9% Nonidet P-40 (Sigma-Aldrich), 20 μl ribonuclease inhibitor and protease inhibitor EDTA-free (Roche). .. We used blocked GammaBind Plus Sepharose beads (GE Healthcare).

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  • 93
    Roche his tagged rnase h2
    <t>RNase</t> H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P
    His Tagged Rnase H2, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 3 article reviews
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    90
    Roche rnase a incubations
    Aub physical interaction with PABP and eIF3d ( A ) CoIP of PABP with GFP-Aub in 0-2 hour-embryos. Wild-type (WT, mock IP) or  nos-Gal4 UASp-GFP-Aub  (GFP IP) embryo extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP and anti-PABP. Inputs are extracts before IP. ( B ) CoIP of Aub with PABP in 0-2 hour-embryos. Wild-type embryo extracts were immunoprecipitated with anti-PABP (PABP IP) or rabbit serum (mock IP), either in the absence or the presence of RNase A. Western blots were revealed with anti-PABP and anti-Aub. Inputs are extracts before IP. ( C ) GST pull-down assays between GST-PABP and HA-Aub. Constructs and interactions are shown in the table. HA-tagged Aub fragments were revealed using western blots with anti-HA. Inputs correspond to 1/10 of in vitro-synthetized HA-Aub fragments before pull-down. GST alone was used as negative control. GST and GST-recombinant proteins used in each pull down are shown in the bottom gel. ( D-E’’ ) Immunostaining of  UASp-GFP-Aub nos-Gal4  embryos with anti-GFP (green) to visualize Aub and anti-PABP (red). Posterior of embryos are shown. Higher magnification showing the distribution of Aub-containing germ granules and PABP foci ( E-E’’ ). Colocalization and overlap between Aub and PABP staining are quantified in   Fig. S3A . The white arrowhead shows PABP foci surrounding a germ granule. Scale bars: 20 μm in  D  and 5 μm in  E . ( F, G ) CoIP of HA-eIF3d with GFP-Aub ( F ) and of Aub with HA-eIF3d ( G ) in 0-2 hour-embryos.  UASp-HA-eIF3d/+; nos-Gal4/+  (mock IP) or  UASp-HA-eIF3d/+; UASp-GFP-Aub nos-Gal4/+  (GFP IP) embryo extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP and anti-HA ( F ). Wild-type (WT, mock IP) or  UASp-HA-eIF3d/+; nos-Gal4/+  (HA IP) embryo extracts were immunoprecipitated with anti-HA, either in the absence or the presence of RNase A. Western blots were revealed with anti-HA and anti-Aub ( G ). Inputs are extracts before IP in  F  and  G . ( H-J’’ ) Immunostaining of  UASp-HA-eIF3d/+; UASp-GFP-Aub nos-Gal4/+  embryos with anti-GFP (green) to visualize Aub and anti-HA (red) to visualize eIF3d. Posterior of embryos are shown. Higher magnification showing the slight accumulation of HA-eIF3d at the posterior pole ( I-I’’ ), and the distribution of Aub-containing germ granules and eIF3d foci ( J-J’’ ). Colocalization and overlap between Aub and eIF3d staining are quantified in   Fig. S3D . The white arrowhead shows eIF3d foci surrounding a germ granule. Scale bars: 20 μm in  H , 10 μm in  I  and 5 μm in  J .
    Rnase A Incubations, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche rnase inhibitor
    Hypoxic response of the PFKFB3 gene in HIF-1α negative cells a ) where exposed to hypoxia for 6 h, and the total <t>RNA</t> was analyzed for PFKFB3 and Glut-1 mRNA by <t>RNase</t> protection assays. b , electrophoretic gel shift assay of nuclear extracts from HIF-1α (+) and HIF-1α (−) cells exposed to normoxia ( N ) or hypoxia ( H ) using a probe ( P ) that contains the erythropoietin HRE. C represents constitutive bands. The rightmost panel shows a supershift assay in hypoxic HIF-1α (+) cells utilizing anti-HIF-1α antibodies.
    Rnase Inhibitor, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 83 article reviews
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    Image Search Results


    RNase H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: RNase H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Chromatin Immunoprecipitation, Proximity Ligation Assay, Irradiation, Imaging

    BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. * P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. * P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Proximity Ligation Assay, Irradiation, Immunoprecipitation, Binding Assay, Recombinant, Real-time Polymerase Chain Reaction, Transfection

    RNase H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: RNase H2 is recruited to DSBs preferentially in the S/G2-phase of the cell cycle. a ChIP of RNASEH2A (top) and γH2AX (bottom) at the AsiSI cut site in uncut or cut DIvA cells. The bar graph shows the fold induction in cut cells (6 h after AsiSI induction) compared to uncut. Error bars represent s.e.m. ( n ≥ 2 biological replicates). b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. Scale bar: 10 μm. c Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) U2OS cells. At least n = 200 cells were counted from four independent experiments. Lines represent mean ± s.e.m. d Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. Scale bar: 10 μm. e Dot plot shows number of signals per nucleus of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells. At least n = 170 cells were counted from three independent experiments. Lines represent mean ± s.e.m. f Representative pictures of super-resolution imaging analysis of γH2AX (cyan) and RNASEH2A (magenta) colocalization in G1- or S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. g Dot plot shows the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in G1- or S-phase cells. At least n = 50 events were counted from three independent experiments. Lines represent mean ± s.e.m. ** P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Chromatin Immunoprecipitation, Proximity Ligation Assay, Irradiation, Imaging

    BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. * P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. * P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Proximity Ligation Assay, Irradiation, Immunoprecipitation, Binding Assay, Recombinant, Real-time Polymerase Chain Reaction, Transfection

    RNase H2 recruitment to DSBs occurs al later time-points after DSB induction. Bar graphs showing fold induction of a γH2AX, b BRCA1, c RPA, d BRCA2, e RNase H2, and f RAD51 signals at the nongenic AsiSI site analyzed in Fig. 1d . Lines represent mean ± s.e.m. ( n ≥ 2 biological replicates). * P

    Journal: Nature Communications

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    doi: 10.1038/s41467-018-07799-2

    Figure Lengend Snippet: RNase H2 recruitment to DSBs occurs al later time-points after DSB induction. Bar graphs showing fold induction of a γH2AX, b BRCA1, c RPA, d BRCA2, e RNase H2, and f RAD51 signals at the nongenic AsiSI site analyzed in Fig. 1d . Lines represent mean ± s.e.m. ( n ≥ 2 biological replicates). * P

    Article Snippet: Briefly, GST-BRCA2 fragments bound to Glutathione–Sepharose beads or biotin-BRC peptides bound to streptavidin beads were incubated with His-tagged RNase H2 in binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, 10 mM NaF, complete protease inhibitor cocktail (Roche)) for 20 min at RT.

    Techniques: Recombinase Polymerase Amplification

    Aub physical interaction with PABP and eIF3d ( A ) CoIP of PABP with GFP-Aub in 0-2 hour-embryos. Wild-type (WT, mock IP) or  nos-Gal4 UASp-GFP-Aub  (GFP IP) embryo extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP and anti-PABP. Inputs are extracts before IP. ( B ) CoIP of Aub with PABP in 0-2 hour-embryos. Wild-type embryo extracts were immunoprecipitated with anti-PABP (PABP IP) or rabbit serum (mock IP), either in the absence or the presence of RNase A. Western blots were revealed with anti-PABP and anti-Aub. Inputs are extracts before IP. ( C ) GST pull-down assays between GST-PABP and HA-Aub. Constructs and interactions are shown in the table. HA-tagged Aub fragments were revealed using western blots with anti-HA. Inputs correspond to 1/10 of in vitro-synthetized HA-Aub fragments before pull-down. GST alone was used as negative control. GST and GST-recombinant proteins used in each pull down are shown in the bottom gel. ( D-E’’ ) Immunostaining of  UASp-GFP-Aub nos-Gal4  embryos with anti-GFP (green) to visualize Aub and anti-PABP (red). Posterior of embryos are shown. Higher magnification showing the distribution of Aub-containing germ granules and PABP foci ( E-E’’ ). Colocalization and overlap between Aub and PABP staining are quantified in   Fig. S3A . The white arrowhead shows PABP foci surrounding a germ granule. Scale bars: 20 μm in  D  and 5 μm in  E . ( F, G ) CoIP of HA-eIF3d with GFP-Aub ( F ) and of Aub with HA-eIF3d ( G ) in 0-2 hour-embryos.  UASp-HA-eIF3d/+; nos-Gal4/+  (mock IP) or  UASp-HA-eIF3d/+; UASp-GFP-Aub nos-Gal4/+  (GFP IP) embryo extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP and anti-HA ( F ). Wild-type (WT, mock IP) or  UASp-HA-eIF3d/+; nos-Gal4/+  (HA IP) embryo extracts were immunoprecipitated with anti-HA, either in the absence or the presence of RNase A. Western blots were revealed with anti-HA and anti-Aub ( G ). Inputs are extracts before IP in  F  and  G . ( H-J’’ ) Immunostaining of  UASp-HA-eIF3d/+; UASp-GFP-Aub nos-Gal4/+  embryos with anti-GFP (green) to visualize Aub and anti-HA (red) to visualize eIF3d. Posterior of embryos are shown. Higher magnification showing the slight accumulation of HA-eIF3d at the posterior pole ( I-I’’ ), and the distribution of Aub-containing germ granules and eIF3d foci ( J-J’’ ). Colocalization and overlap between Aub and eIF3d staining are quantified in   Fig. S3D . The white arrowhead shows eIF3d foci surrounding a germ granule. Scale bars: 20 μm in  H , 10 μm in  I  and 5 μm in  J .

    Journal: bioRxiv

    Article Title: The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm

    doi: 10.1101/859561

    Figure Lengend Snippet: Aub physical interaction with PABP and eIF3d ( A ) CoIP of PABP with GFP-Aub in 0-2 hour-embryos. Wild-type (WT, mock IP) or nos-Gal4 UASp-GFP-Aub (GFP IP) embryo extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP and anti-PABP. Inputs are extracts before IP. ( B ) CoIP of Aub with PABP in 0-2 hour-embryos. Wild-type embryo extracts were immunoprecipitated with anti-PABP (PABP IP) or rabbit serum (mock IP), either in the absence or the presence of RNase A. Western blots were revealed with anti-PABP and anti-Aub. Inputs are extracts before IP. ( C ) GST pull-down assays between GST-PABP and HA-Aub. Constructs and interactions are shown in the table. HA-tagged Aub fragments were revealed using western blots with anti-HA. Inputs correspond to 1/10 of in vitro-synthetized HA-Aub fragments before pull-down. GST alone was used as negative control. GST and GST-recombinant proteins used in each pull down are shown in the bottom gel. ( D-E’’ ) Immunostaining of UASp-GFP-Aub nos-Gal4 embryos with anti-GFP (green) to visualize Aub and anti-PABP (red). Posterior of embryos are shown. Higher magnification showing the distribution of Aub-containing germ granules and PABP foci ( E-E’’ ). Colocalization and overlap between Aub and PABP staining are quantified in Fig. S3A . The white arrowhead shows PABP foci surrounding a germ granule. Scale bars: 20 μm in D and 5 μm in E . ( F, G ) CoIP of HA-eIF3d with GFP-Aub ( F ) and of Aub with HA-eIF3d ( G ) in 0-2 hour-embryos. UASp-HA-eIF3d/+; nos-Gal4/+ (mock IP) or UASp-HA-eIF3d/+; UASp-GFP-Aub nos-Gal4/+ (GFP IP) embryo extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP and anti-HA ( F ). Wild-type (WT, mock IP) or UASp-HA-eIF3d/+; nos-Gal4/+ (HA IP) embryo extracts were immunoprecipitated with anti-HA, either in the absence or the presence of RNase A. Western blots were revealed with anti-HA and anti-Aub ( G ). Inputs are extracts before IP in F and G . ( H-J’’ ) Immunostaining of UASp-HA-eIF3d/+; UASp-GFP-Aub nos-Gal4/+ embryos with anti-GFP (green) to visualize Aub and anti-HA (red) to visualize eIF3d. Posterior of embryos are shown. Higher magnification showing the slight accumulation of HA-eIF3d at the posterior pole ( I-I’’ ), and the distribution of Aub-containing germ granules and eIF3d foci ( J-J’’ ). Colocalization and overlap between Aub and eIF3d staining are quantified in Fig. S3D . The white arrowhead shows eIF3d foci surrounding a germ granule. Scale bars: 20 μm in H , 10 μm in I and 5 μm in J .

    Article Snippet: HA-Aub proteins were synthesized in vitro using the TnT Coupled reticulocyte lysate system (Promega), and were incubated with immobilized GST fusion proteins in 400 µl binding buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 0.2 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40, cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Roche)) containing 0.2 µg µL−1 RNase A. Incubations were performed for 1 h at 4 °C followed by 30 min at room temperature.

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Construct, In Vitro, Negative Control, Recombinant, Immunostaining, Staining

    Hypoxic response of the PFKFB3 gene in HIF-1α negative cells a ) where exposed to hypoxia for 6 h, and the total RNA was analyzed for PFKFB3 and Glut-1 mRNA by RNase protection assays. b , electrophoretic gel shift assay of nuclear extracts from HIF-1α (+) and HIF-1α (−) cells exposed to normoxia ( N ) or hypoxia ( H ) using a probe ( P ) that contains the erythropoietin HRE. C represents constitutive bands. The rightmost panel shows a supershift assay in hypoxic HIF-1α (+) cells utilizing anti-HIF-1α antibodies.

    Journal: The Journal of biological chemistry

    Article Title: Hypoxia-inducible Factor-1-mediated Expression of the 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) Gene

    doi: 10.1074/jbc.M110978200

    Figure Lengend Snippet: Hypoxic response of the PFKFB3 gene in HIF-1α negative cells a ) where exposed to hypoxia for 6 h, and the total RNA was analyzed for PFKFB3 and Glut-1 mRNA by RNase protection assays. b , electrophoretic gel shift assay of nuclear extracts from HIF-1α (+) and HIF-1α (−) cells exposed to normoxia ( N ) or hypoxia ( H ) using a probe ( P ) that contains the erythropoietin HRE. C represents constitutive bands. The rightmost panel shows a supershift assay in hypoxic HIF-1α (+) cells utilizing anti-HIF-1α antibodies.

    Article Snippet: T3 and T7 RNA polymerases, RNase inhibitor, and DNase I (Rnase free) where from Roche Molecular Biochemicals.

    Techniques: Electrophoretic Mobility Shift Assay

    Expression of the PFKFB3 gene in pVHL-deficient cells a , total RNA from normoxic VHL deficient (−) renal carcinoma 786-0 cells and their VHL positive (+) controls where analyzed for PFKFB3, Glut-1, and VEGF mRNA expression by RNase protection assays. b , whole cell extracts from VHL (+) and VHL (−) were analyzed by Western blots using anti-HIF-2α and anti-VHL antibodies.

    Journal: The Journal of biological chemistry

    Article Title: Hypoxia-inducible Factor-1-mediated Expression of the 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) Gene

    doi: 10.1074/jbc.M110978200

    Figure Lengend Snippet: Expression of the PFKFB3 gene in pVHL-deficient cells a , total RNA from normoxic VHL deficient (−) renal carcinoma 786-0 cells and their VHL positive (+) controls where analyzed for PFKFB3, Glut-1, and VEGF mRNA expression by RNase protection assays. b , whole cell extracts from VHL (+) and VHL (−) were analyzed by Western blots using anti-HIF-2α and anti-VHL antibodies.

    Article Snippet: T3 and T7 RNA polymerases, RNase inhibitor, and DNase I (Rnase free) where from Roche Molecular Biochemicals.

    Techniques: Expressing, Western Blot