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  • 99
    Thermo Fisher mouse anti brutp
    Bleb-associated genetic regions are actively transcribed. ( A ) Primary p.S143F fibroblasts (p33) were stained for either RNA polymerase II (first row) or active RNA polymerase II (Pol IIo, second row), LA and <t>LB1.</t> Hoechst was used to visualize DNA. Single mid-plane confocal sections from the whole cell are shown. RNA polymerase II and active RNA polymerase II (Pol IIo) are enriched in the blebs (arrows). Scale bars 5 μm. ( B ) Transcriptional activity in p.S143F fibroblasts (p30) was visualized by <t>BrUTP</t> incorporation into nascent RNA by scratch labeling. DAPI was used to visualize DNA. A single mid-plane confocal section is shown. Note the intense BrUTP staining in the bleb compartment (arrow). Scale bar 5 μm. ( C ) p.S143F fibroblasts (p36) were subjected to RNA FISH with a probe for a highly transcribed region on chromosome 19. DAPI was used to visualize DNA. Single confocal sections from mid-plane and nuclear surface are shown. FISH signals are detectable in the bleb region (arrow). Scale bar 5 μm. ( D ) p.S143F fibroblasts (p21) were stained for LA/C and histone H3 trimethylated at lysine 4 or lysine 36. Hoechst was used to visualize DNA. Single mid-plane confocal sections are shown. Both histone marks are detectable in the bleb region (arrows). Scale bars 5 μm.
    Mouse Anti Brutp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore br utp
    p68 mutation blocks gene shutoff during heat shock. ( A–D ) Control ( A,C ) and CB02119/Lip H ( B,D ) larvae were heat-shocked at 37°C for 20 min and immediately processed. The cells were stained using anti-H5 antibodies (green) and anti-HSF antibodies (red) (cf. C and D ). Examining the anti-H5 staining alone reveals that active Pol II is limited to major heat-shock puffs in control cells ( C ), while in p68 mutant cells, it is present at heat-shock loci, other discrete sites, and generally along chromosome arms ( D ) ( hsp70 gene loci at 87A and 87C are indicated). ( E–F ) Control ( E,G ) and CB02119/Lip H ( F,H ) larvae were heat-shocked at 37°C for 10 min, pulse-labeled with <t>Br-UTP,</t> and heat-shocked for an additional 20 min. ( E,G ) Control samples exhibit Br-UTP labeling only at heat-shock response puffs. ( F,H ) p68 mutant cells show high levels of Br-UTP incorporation at many chromosomal sites including the nucleolus, demonstrating that a broad number of genes continue to transcribe <t>RNA</t> during heat shock in the absence of p68.
    Br Utp, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Boehringer Mannheim br utp
    p68 mutation blocks gene shutoff during heat shock. ( A–D ) Control ( A,C ) and CB02119/Lip H ( B,D ) larvae were heat-shocked at 37°C for 20 min and immediately processed. The cells were stained using anti-H5 antibodies (green) and anti-HSF antibodies (red) (cf. C and D ). Examining the anti-H5 staining alone reveals that active Pol II is limited to major heat-shock puffs in control cells ( C ), while in p68 mutant cells, it is present at heat-shock loci, other discrete sites, and generally along chromosome arms ( D ) ( hsp70 gene loci at 87A and 87C are indicated). ( E–F ) Control ( E,G ) and CB02119/Lip H ( F,H ) larvae were heat-shocked at 37°C for 10 min, pulse-labeled with <t>Br-UTP,</t> and heat-shocked for an additional 20 min. ( E,G ) Control samples exhibit Br-UTP labeling only at heat-shock response puffs. ( F,H ) p68 mutant cells show high levels of Br-UTP incorporation at many chromosomal sites including the nucleolus, demonstrating that a broad number of genes continue to transcribe <t>RNA</t> during heat shock in the absence of p68.
    Br Utp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology br utp
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Br Utp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology br utp beads
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Br Utp Beads, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher m br utp
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    M Br Utp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher bromo utp br utp
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Bromo Utp Br Utp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher br utp
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Br Utp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti br utp beads
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Anti Br Utp Beads, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche bromo utp br utp
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Bromo Utp Br Utp, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti br utp agarose beads
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Anti Br Utp Agarose Beads, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore 5 bromo utp br utp
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    5 Bromo Utp Br Utp, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche anti br utp antibody
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Anti Br Utp Antibody, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology br utp specific antibody
    Effect of <t>pCU</t> on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with <t>BrUTP</t> at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.
    Br Utp Specific Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti br utp mab
    ActD at a concentration of 0.04 μg/ml selectively inhibits Pol I transcription without significantly affecting other polymerases and general nuclear organization. A and A′ show the in situ incorporation of <t>Br-UTP,</t> and B and B′ show the same cells corresponding to those in A and A′ immunolabeled with anti-fibrillarin antibody. Daughter cells grown in the presence of ActD form mininucleoli that are immunolabeled with anti-fibrillarin antibody (B, arrows). They do not incorporate Br-UTP (A, arrows), whereas untreated nucleoli (B′, arrows) actively incorporate Br-UTP after pulse labeling (A′). Although Br-UTP incorporation in nucleoli declines in treated cells, the nucleoplasmic Br-UTP incorporation from Pol II and Pol III transcription is not altered (A). The speckled distribution of SC35 (C) and the predominantly nuclear localization of <t>hnRNP</t> A1 (D) are also not changed significantly in treated cells. Bar, 10 μm.
    Anti Br Utp Mab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore triphosphate sodium salt br utp sigma 100 mm in 2 mm pipes buffer
    ActD at a concentration of 0.04 μg/ml selectively inhibits Pol I transcription without significantly affecting other polymerases and general nuclear organization. A and A′ show the in situ incorporation of <t>Br-UTP,</t> and B and B′ show the same cells corresponding to those in A and A′ immunolabeled with anti-fibrillarin antibody. Daughter cells grown in the presence of ActD form mininucleoli that are immunolabeled with anti-fibrillarin antibody (B, arrows). They do not incorporate Br-UTP (A, arrows), whereas untreated nucleoli (B′, arrows) actively incorporate Br-UTP after pulse labeling (A′). Although Br-UTP incorporation in nucleoli declines in treated cells, the nucleoplasmic Br-UTP incorporation from Pol II and Pol III transcription is not altered (A). The speckled distribution of SC35 (C) and the predominantly nuclear localization of <t>hnRNP</t> A1 (D) are also not changed significantly in treated cells. Bar, 10 μm.
    Triphosphate Sodium Salt Br Utp Sigma 100 Mm In 2 Mm Pipes Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore brutp
    Newly synthesized viral RNAs are present inside the viral IBs. (A) BSR cells were infected at an MOI of 3 for 16 h p.i., transfected with <t>BrUTP</t> for 20 min in the presence <t>(+ActD)</t> or absence (-ActD) of ActD. P protein (as a marker of the Negri bodies) was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-goat anti-rabbit IgG (P). Newly synthesized RNAs were detected by using mouse MAb anti-BrdU antibody (BrUTP) and goat anti-mouse Alexa 488. Colocalization of the viral RNA (vRNA) with the IBs is shown in the merged panel in the presence of the drug (+ActD). In cells untreated with ActD, cellular mRNA is produced in vast excess over vRNA. Thus, for these images, the PMT gain of the confocal laser scanning microscope is lower, so the signal for vRNA is lower than for that shown in the presence of the drug, and specific colocalization is not readily observed. The scale bars correspond to 12 μm. (B) BrUTP signals are specific to RNA produced by rabies virus. BSR cells were infected with the VTF7-3 recombinant virus and cotransfected with plasmids expressing N and P proteins. Cells were then transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein and RNA were detected as in panel A. Note that in the absence of the drug, no BrUTP signal is detectable in the IBs formed by N and P proteins, but some RNA signals are detected in the cytoplasmic inclusions, most probably due to vaccinia virus infection (-ActD). In the presence of the drug, no RNA labeling is observed (+ActD).
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    brutp  (Roche)
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    (a) Subcellular localization of the 16-kDa protein. (A) Cos-7 cells overexpressing pEGFP-16k at 16 h posttransfection showing perinuclear staining. (B) Cos-7 cells overexpressing the EGFP control vector (pEGFP) showing diffuse staining. (C) Immunofluorescent staining of <t>IBV-infected</t> Vero cells with anti-16-kDa protein serum (1:20) at 13 h postinfection showing distinct punctate perinuclear staining. (D) Immunofluorescent staining of mock-infected Vero cells with anti-16-kDa protein serum (1:20) at 13 h postinfection. (E) Double labeling of IBV-infected Vero cells that were transfected with <t>BrUTP</t> at 10 h postinfection showing the staining profile of anti-16-kDa protein serum. (F) Double labeling of IBV-infected Vero cells that were transfected with BrUTP at 10 h postinfection showing the staining pattern of anti-BrUTP serum. (G) Superimposition of images E and F. All images were taken from a Zeiss Axioplan confocal microscope. (b) Membrane association of the 16-kDa protein. Cos-7 cells expressing the 16-kDa protein were labeled with [ 35 S]methionine-cysteine for 4 h and harvested. Cells were lysed with a Dounce homogenizer and fractionated into membrane (M) and cytosol (C) fractions at pH 7 (lanes 1 and 2) by ultracentrifugation. Polypeptides were immunoprecipitated with anti-16-kDa protein serum, separated on an SDS-15% polyacrylamide gel, and detected by fluorography. Membrane (M) pellets were resuspended in hypotonic buffer; treated with 1% Triton X-100 (TX-100), 100 mM Na 2 CO 3 , or 1 M KCl; and further fractionated into supernatant (S) and pellet (P) fractions by ultracentrifugation. Polypeptides were immunoprecipitated with anti-16-kDa protein serum, separated on an SDS-15% polyacrylamide gel, and detected by fluorography.
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    (A) NF-Y is associated with active transcription sites in living cells. After in vivo incorporation of <t>BrUTP</t> (run-on), cells were fixed and endogenous NF-YA (ii) and nascent RNA transcripts (i) were detected by indirect immunofluorescence combined with Confocal Scanning Laser Microscopy by using anti-NF-YA and anti-BrU antibodies. In the overlay (iii), yellow indicates colocalizations between NF-YA (green) and transcription sites (red). In panels vi and x cells were immunostained with anti-NF-YA, in panels v and ix with <t>anti-RPII</t> CTD repeat YSPTSPS (phospho S2) and anti-total RPII respectively. The majority of NF-YA (red) colocalizes with the activated form of RPII (green)(vii). (B) Cells were immunostained with anti-NF-YA (vii-xii), -acetylated H3K9 (i), -acetylated H4 (ii), -tri-methylated H3K9 (iii), -di-methylated H3K27 (iv), -tri-methylated H4K20 (v) and -NF-YB (vi) antibodies. The majority of NF-YA colocalizes with acetylated (xiii, xiv), but poor colocalization occurs with methylated histones (xv, xvi,xvii). Panel xviii shows the overlay of two subunits of NF-Y, NF-YA (xii) and NF-YB (vi). Panels from xix to xxiv represent a typical optical field of the merge. In figure 1A and 1B confocal analysis of single optical section is shown. The images have been collected with a 60x objective.
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    (A) NF-Y is associated with active transcription sites in living cells. After in vivo incorporation of <t>BrUTP</t> (run-on), cells were fixed and endogenous NF-YA (ii) and nascent RNA transcripts (i) were detected by indirect immunofluorescence combined with Confocal Scanning Laser Microscopy by using anti-NF-YA and anti-BrU antibodies. In the overlay (iii), yellow indicates colocalizations between NF-YA (green) and transcription sites (red). In panels vi and x cells were immunostained with anti-NF-YA, in panels v and ix with <t>anti-RPII</t> CTD repeat YSPTSPS (phospho S2) and anti-total RPII respectively. The majority of NF-YA (red) colocalizes with the activated form of RPII (green)(vii). (B) Cells were immunostained with anti-NF-YA (vii-xii), -acetylated H3K9 (i), -acetylated H4 (ii), -tri-methylated H3K9 (iii), -di-methylated H3K27 (iv), -tri-methylated H4K20 (v) and -NF-YB (vi) antibodies. The majority of NF-YA colocalizes with acetylated (xiii, xiv), but poor colocalization occurs with methylated histones (xv, xvi,xvii). Panel xviii shows the overlay of two subunits of NF-Y, NF-YA (xii) and NF-YB (vi). Panels from xix to xxiv represent a typical optical field of the merge. In figure 1A and 1B confocal analysis of single optical section is shown. The images have been collected with a 60x objective.
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    (A) NF-Y is associated with active transcription sites in living cells. After in vivo incorporation of <t>BrUTP</t> (run-on), cells were fixed and endogenous NF-YA (ii) and nascent RNA transcripts (i) were detected by indirect immunofluorescence combined with Confocal Scanning Laser Microscopy by using anti-NF-YA and anti-BrU antibodies. In the overlay (iii), yellow indicates colocalizations between NF-YA (green) and transcription sites (red). In panels vi and x cells were immunostained with anti-NF-YA, in panels v and ix with <t>anti-RPII</t> CTD repeat YSPTSPS (phospho S2) and anti-total RPII respectively. The majority of NF-YA (red) colocalizes with the activated form of RPII (green)(vii). (B) Cells were immunostained with anti-NF-YA (vii-xii), -acetylated H3K9 (i), -acetylated H4 (ii), -tri-methylated H3K9 (iii), -di-methylated H3K27 (iv), -tri-methylated H4K20 (v) and -NF-YB (vi) antibodies. The majority of NF-YA colocalizes with acetylated (xiii, xiv), but poor colocalization occurs with methylated histones (xv, xvi,xvii). Panel xviii shows the overlay of two subunits of NF-Y, NF-YA (xii) and NF-YB (vi). Panels from xix to xxiv represent a typical optical field of the merge. In figure 1A and 1B confocal analysis of single optical section is shown. The images have been collected with a 60x objective.
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    (A) NF-Y is associated with active transcription sites in living cells. After in vivo incorporation of <t>BrUTP</t> (run-on), cells were fixed and endogenous NF-YA (ii) and nascent RNA transcripts (i) were detected by indirect immunofluorescence combined with Confocal Scanning Laser Microscopy by using anti-NF-YA and anti-BrU antibodies. In the overlay (iii), yellow indicates colocalizations between NF-YA (green) and transcription sites (red). In panels vi and x cells were immunostained with anti-NF-YA, in panels v and ix with <t>anti-RPII</t> CTD repeat YSPTSPS (phospho S2) and anti-total RPII respectively. The majority of NF-YA (red) colocalizes with the activated form of RPII (green)(vii). (B) Cells were immunostained with anti-NF-YA (vii-xii), -acetylated H3K9 (i), -acetylated H4 (ii), -tri-methylated H3K9 (iii), -di-methylated H3K27 (iv), -tri-methylated H4K20 (v) and -NF-YB (vi) antibodies. The majority of NF-YA colocalizes with acetylated (xiii, xiv), but poor colocalization occurs with methylated histones (xv, xvi,xvii). Panel xviii shows the overlay of two subunits of NF-Y, NF-YA (xii) and NF-YB (vi). Panels from xix to xxiv represent a typical optical field of the merge. In figure 1A and 1B confocal analysis of single optical section is shown. The images have been collected with a 60x objective.
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    Image Search Results


    Bleb-associated genetic regions are actively transcribed. ( A ) Primary p.S143F fibroblasts (p33) were stained for either RNA polymerase II (first row) or active RNA polymerase II (Pol IIo, second row), LA and LB1. Hoechst was used to visualize DNA. Single mid-plane confocal sections from the whole cell are shown. RNA polymerase II and active RNA polymerase II (Pol IIo) are enriched in the blebs (arrows). Scale bars 5 μm. ( B ) Transcriptional activity in p.S143F fibroblasts (p30) was visualized by BrUTP incorporation into nascent RNA by scratch labeling. DAPI was used to visualize DNA. A single mid-plane confocal section is shown. Note the intense BrUTP staining in the bleb compartment (arrow). Scale bar 5 μm. ( C ) p.S143F fibroblasts (p36) were subjected to RNA FISH with a probe for a highly transcribed region on chromosome 19. DAPI was used to visualize DNA. Single confocal sections from mid-plane and nuclear surface are shown. FISH signals are detectable in the bleb region (arrow). Scale bar 5 μm. ( D ) p.S143F fibroblasts (p21) were stained for LA/C and histone H3 trimethylated at lysine 4 or lysine 36. Hoechst was used to visualize DNA. Single mid-plane confocal sections are shown. Both histone marks are detectable in the bleb region (arrows). Scale bars 5 μm.

    Journal: Nucleus

    Article Title: Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells

    doi: 10.1080/19491034.2015.1004256

    Figure Lengend Snippet: Bleb-associated genetic regions are actively transcribed. ( A ) Primary p.S143F fibroblasts (p33) were stained for either RNA polymerase II (first row) or active RNA polymerase II (Pol IIo, second row), LA and LB1. Hoechst was used to visualize DNA. Single mid-plane confocal sections from the whole cell are shown. RNA polymerase II and active RNA polymerase II (Pol IIo) are enriched in the blebs (arrows). Scale bars 5 μm. ( B ) Transcriptional activity in p.S143F fibroblasts (p30) was visualized by BrUTP incorporation into nascent RNA by scratch labeling. DAPI was used to visualize DNA. A single mid-plane confocal section is shown. Note the intense BrUTP staining in the bleb compartment (arrow). Scale bar 5 μm. ( C ) p.S143F fibroblasts (p36) were subjected to RNA FISH with a probe for a highly transcribed region on chromosome 19. DAPI was used to visualize DNA. Single confocal sections from mid-plane and nuclear surface are shown. FISH signals are detectable in the bleb region (arrow). Scale bar 5 μm. ( D ) p.S143F fibroblasts (p21) were stained for LA/C and histone H3 trimethylated at lysine 4 or lysine 36. Hoechst was used to visualize DNA. Single mid-plane confocal sections are shown. Both histone marks are detectable in the bleb region (arrows). Scale bars 5 μm.

    Article Snippet: Primary antibodies used were mouse monoclonal anti-LA/C (1:300; JoL2, Chemicon, cat.# MAB3211), rabbit polyclonal anti-LA (1:1000) , rabbit polyclonal anti-LB1 (1:1000) , mouse monoclonal anti-LB2 (1:100; LN43, Abcam, cat.# ab8983), rabbit polyclonal anti-H3K9me3 (1:1000, Abcam, cat.# ab8898) and rabbit polyclonal anti-H3K27me3 (1:1000, Abcam, cat.# ab108245), rabbit polyclonal anti-H3K4me2 (1:1000, Abcam, cat.# ab7766), rabbit polyclonal anti-H3K36me3 (1:1000, Abcam, cat.# ab9050, rabbit polyclonal anti-AcH3 (1:1000, Upstate/Merck Millipore, cat.# 06–599), mouse monoclonal anti-Pol II (1:1000, 4H8, Abcam, cat.# ab5408), mouse monoclonal anti-Pol II phosphorylated at Ser2 (H5, 1:100, Abcam, cat.# ab24758), mouse monoclonal anti-FLAG (1:400, clone M2, Sigma, cat.# F1804), rabbit polyclonal anti-SUN1 and anti-SUN2 (1:40 and 1:20; kindly provided by Didier Hodzic) , goat polyclonal anti-LB1 (1:200 Santa Cruz cat.# sc-6217), mouse anti-BrUTP (1:100, Caltag Laboratories, clone Br-3), rabbit polyclonal anti-p53 (1:100, Imgenex, cat.# IMG-533) and rabbit polyclonal anti-SKIP (NCOA62) (1:100, Abcam, cat.# ab153887).

    Techniques: Staining, Activity Assay, Labeling, Fluorescence In Situ Hybridization

    p68 mutation blocks gene shutoff during heat shock. ( A–D ) Control ( A,C ) and CB02119/Lip H ( B,D ) larvae were heat-shocked at 37°C for 20 min and immediately processed. The cells were stained using anti-H5 antibodies (green) and anti-HSF antibodies (red) (cf. C and D ). Examining the anti-H5 staining alone reveals that active Pol II is limited to major heat-shock puffs in control cells ( C ), while in p68 mutant cells, it is present at heat-shock loci, other discrete sites, and generally along chromosome arms ( D ) ( hsp70 gene loci at 87A and 87C are indicated). ( E–F ) Control ( E,G ) and CB02119/Lip H ( F,H ) larvae were heat-shocked at 37°C for 10 min, pulse-labeled with Br-UTP, and heat-shocked for an additional 20 min. ( E,G ) Control samples exhibit Br-UTP labeling only at heat-shock response puffs. ( F,H ) p68 mutant cells show high levels of Br-UTP incorporation at many chromosomal sites including the nucleolus, demonstrating that a broad number of genes continue to transcribe RNA during heat shock in the absence of p68.

    Journal: Genes & Development

    Article Title: The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin

    doi: 10.1101/gad.1396306

    Figure Lengend Snippet: p68 mutation blocks gene shutoff during heat shock. ( A–D ) Control ( A,C ) and CB02119/Lip H ( B,D ) larvae were heat-shocked at 37°C for 20 min and immediately processed. The cells were stained using anti-H5 antibodies (green) and anti-HSF antibodies (red) (cf. C and D ). Examining the anti-H5 staining alone reveals that active Pol II is limited to major heat-shock puffs in control cells ( C ), while in p68 mutant cells, it is present at heat-shock loci, other discrete sites, and generally along chromosome arms ( D ) ( hsp70 gene loci at 87A and 87C are indicated). ( E–F ) Control ( E,G ) and CB02119/Lip H ( F,H ) larvae were heat-shocked at 37°C for 10 min, pulse-labeled with Br-UTP, and heat-shocked for an additional 20 min. ( E,G ) Control samples exhibit Br-UTP labeling only at heat-shock response puffs. ( F,H ) p68 mutant cells show high levels of Br-UTP incorporation at many chromosomal sites including the nucleolus, demonstrating that a broad number of genes continue to transcribe RNA during heat shock in the absence of p68.

    Article Snippet: A final concentration of 2 mM Br-UTP (Sigma) was used to pulse-label nascent RNA in salivary glands.

    Techniques: Mutagenesis, Staining, Labeling

    p68 mutants display RNA export defects. ( A,B ) Control ( A ) and CB02119/Lip F ( B ) transheterozygotes labeled for hsp70 RNA immediately after a 20-min heat shock. The edge of representative nuclei is outlined in white (boxes). p68 mutant salivary glands have less cytoplasmic hsp70 RNA and qualitatively higher levels of hsp70 RNA at transcription sites. ( C,D ) Control ( C ) and CB02119/Lip F ( D ) salivary glands labeled with Br-UTP. Control cells exhibit cytoplasmic staining and punctuate nuclear Br-UTP staining, whereas p68 mutant salivary glands display robust Br-UTP labeling on polytene chromosomal bands. Little signal was observed in the cytoplasm of mutant cells. ( E–H ) Control ( E,G ) and CB02119/Lip F ( F,H ) salivary gland cells stained with anti-SBR (NXF1) antibodies ( E,F ) or anti-ALY (REF1) antibodies ( G,H ). SBR and ALY are found along the nuclear periphery and in the nucleoplasm of control cells. In p68 mutant cells, their distribution is changed, and both export factors accumulate in the nuclear interior.

    Journal: Genes & Development

    Article Title: The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin

    doi: 10.1101/gad.1396306

    Figure Lengend Snippet: p68 mutants display RNA export defects. ( A,B ) Control ( A ) and CB02119/Lip F ( B ) transheterozygotes labeled for hsp70 RNA immediately after a 20-min heat shock. The edge of representative nuclei is outlined in white (boxes). p68 mutant salivary glands have less cytoplasmic hsp70 RNA and qualitatively higher levels of hsp70 RNA at transcription sites. ( C,D ) Control ( C ) and CB02119/Lip F ( D ) salivary glands labeled with Br-UTP. Control cells exhibit cytoplasmic staining and punctuate nuclear Br-UTP staining, whereas p68 mutant salivary glands display robust Br-UTP labeling on polytene chromosomal bands. Little signal was observed in the cytoplasm of mutant cells. ( E–H ) Control ( E,G ) and CB02119/Lip F ( F,H ) salivary gland cells stained with anti-SBR (NXF1) antibodies ( E,F ) or anti-ALY (REF1) antibodies ( G,H ). SBR and ALY are found along the nuclear periphery and in the nucleoplasm of control cells. In p68 mutant cells, their distribution is changed, and both export factors accumulate in the nuclear interior.

    Article Snippet: A final concentration of 2 mM Br-UTP (Sigma) was used to pulse-label nascent RNA in salivary glands.

    Techniques: Labeling, Mutagenesis, Staining

    Effect of pCU on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with BrUTP at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.

    Journal: Neuro-Oncology

    Article Title: uPAR and cathepsin B inhibition enhanced radiation-induced apoptosis in gliomainitiating cells

    doi: 10.1093/neuonc/nos088

    Figure Lengend Snippet: Effect of pCU on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with BrUTP at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.

    Article Snippet: Here, GICs and non-GICs were plated onto 4-well chamber slides and treated with pCU and radiation as described previously, and 150 µL of medium containing BrUTP (SC: 70443, Santa Cruz Biotechnology) at a final concentration of 5 mM were added to each well.

    Techniques: Transfection, Incubation, Concentration Assay, Staining, Microscopy, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    ActD at a concentration of 0.04 μg/ml selectively inhibits Pol I transcription without significantly affecting other polymerases and general nuclear organization. A and A′ show the in situ incorporation of Br-UTP, and B and B′ show the same cells corresponding to those in A and A′ immunolabeled with anti-fibrillarin antibody. Daughter cells grown in the presence of ActD form mininucleoli that are immunolabeled with anti-fibrillarin antibody (B, arrows). They do not incorporate Br-UTP (A, arrows), whereas untreated nucleoli (B′, arrows) actively incorporate Br-UTP after pulse labeling (A′). Although Br-UTP incorporation in nucleoli declines in treated cells, the nucleoplasmic Br-UTP incorporation from Pol II and Pol III transcription is not altered (A). The speckled distribution of SC35 (C) and the predominantly nuclear localization of hnRNP A1 (D) are also not changed significantly in treated cells. Bar, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Initiation of Nucleolar Assembly Is Independent of RNA Polymerase I Transcription

    doi:

    Figure Lengend Snippet: ActD at a concentration of 0.04 μg/ml selectively inhibits Pol I transcription without significantly affecting other polymerases and general nuclear organization. A and A′ show the in situ incorporation of Br-UTP, and B and B′ show the same cells corresponding to those in A and A′ immunolabeled with anti-fibrillarin antibody. Daughter cells grown in the presence of ActD form mininucleoli that are immunolabeled with anti-fibrillarin antibody (B, arrows). They do not incorporate Br-UTP (A, arrows), whereas untreated nucleoli (B′, arrows) actively incorporate Br-UTP after pulse labeling (A′). Although Br-UTP incorporation in nucleoli declines in treated cells, the nucleoplasmic Br-UTP incorporation from Pol II and Pol III transcription is not altered (A). The speckled distribution of SC35 (C) and the predominantly nuclear localization of hnRNP A1 (D) are also not changed significantly in treated cells. Bar, 10 μm.

    Article Snippet: Antibodies used in this study included anti-UBF–specific human serum at a dilution of 1:300 ( ; ), anti-nucleolin mAb at a dilution of 1:3000 , anti-fibrillarin human serum at a dilution of 1:100 , anti-SC35 at a dilution of 1:1000 , anti-hnRNP A1 at a dilution of 1:300 , and anti-Br-UTP mAb at a dilution of 1:500 (Sigma Chemical, St. Louis, MO) for 1 h at room temperature.

    Techniques: Concentration Assay, In Situ, Immunolabeling, Labeling

    Newly synthesized viral RNAs are present inside the viral IBs. (A) BSR cells were infected at an MOI of 3 for 16 h p.i., transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein (as a marker of the Negri bodies) was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-goat anti-rabbit IgG (P). Newly synthesized RNAs were detected by using mouse MAb anti-BrdU antibody (BrUTP) and goat anti-mouse Alexa 488. Colocalization of the viral RNA (vRNA) with the IBs is shown in the merged panel in the presence of the drug (+ActD). In cells untreated with ActD, cellular mRNA is produced in vast excess over vRNA. Thus, for these images, the PMT gain of the confocal laser scanning microscope is lower, so the signal for vRNA is lower than for that shown in the presence of the drug, and specific colocalization is not readily observed. The scale bars correspond to 12 μm. (B) BrUTP signals are specific to RNA produced by rabies virus. BSR cells were infected with the VTF7-3 recombinant virus and cotransfected with plasmids expressing N and P proteins. Cells were then transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein and RNA were detected as in panel A. Note that in the absence of the drug, no BrUTP signal is detectable in the IBs formed by N and P proteins, but some RNA signals are detected in the cytoplasmic inclusions, most probably due to vaccinia virus infection (-ActD). In the presence of the drug, no RNA labeling is observed (+ActD).

    Journal: Journal of Virology

    Article Title: Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication ▿

    doi: 10.1128/JVI.00554-09

    Figure Lengend Snippet: Newly synthesized viral RNAs are present inside the viral IBs. (A) BSR cells were infected at an MOI of 3 for 16 h p.i., transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein (as a marker of the Negri bodies) was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-goat anti-rabbit IgG (P). Newly synthesized RNAs were detected by using mouse MAb anti-BrdU antibody (BrUTP) and goat anti-mouse Alexa 488. Colocalization of the viral RNA (vRNA) with the IBs is shown in the merged panel in the presence of the drug (+ActD). In cells untreated with ActD, cellular mRNA is produced in vast excess over vRNA. Thus, for these images, the PMT gain of the confocal laser scanning microscope is lower, so the signal for vRNA is lower than for that shown in the presence of the drug, and specific colocalization is not readily observed. The scale bars correspond to 12 μm. (B) BrUTP signals are specific to RNA produced by rabies virus. BSR cells were infected with the VTF7-3 recombinant virus and cotransfected with plasmids expressing N and P proteins. Cells were then transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein and RNA were detected as in panel A. Note that in the absence of the drug, no BrUTP signal is detectable in the IBs formed by N and P proteins, but some RNA signals are detected in the cytoplasmic inclusions, most probably due to vaccinia virus infection (-ActD). In the presence of the drug, no RNA labeling is observed (+ActD).

    Article Snippet: Briefly, the infected cells (16 h p.i.) were treated with 15 μg of ActD/ml for 1 h and then transfected with BrUTP (B 7166; Sigma) at a final concentration of 10 mM by using Lipofectamine 2000 (Invitrogen) and maintained in the presence of ActD for 20 min.

    Techniques: Synthesized, Infection, Transfection, Marker, Incubation, Produced, Laser-Scanning Microscopy, Recombinant, Expressing, Labeling

    (a) Subcellular localization of the 16-kDa protein. (A) Cos-7 cells overexpressing pEGFP-16k at 16 h posttransfection showing perinuclear staining. (B) Cos-7 cells overexpressing the EGFP control vector (pEGFP) showing diffuse staining. (C) Immunofluorescent staining of IBV-infected Vero cells with anti-16-kDa protein serum (1:20) at 13 h postinfection showing distinct punctate perinuclear staining. (D) Immunofluorescent staining of mock-infected Vero cells with anti-16-kDa protein serum (1:20) at 13 h postinfection. (E) Double labeling of IBV-infected Vero cells that were transfected with BrUTP at 10 h postinfection showing the staining profile of anti-16-kDa protein serum. (F) Double labeling of IBV-infected Vero cells that were transfected with BrUTP at 10 h postinfection showing the staining pattern of anti-BrUTP serum. (G) Superimposition of images E and F. All images were taken from a Zeiss Axioplan confocal microscope. (b) Membrane association of the 16-kDa protein. Cos-7 cells expressing the 16-kDa protein were labeled with [ 35 S]methionine-cysteine for 4 h and harvested. Cells were lysed with a Dounce homogenizer and fractionated into membrane (M) and cytosol (C) fractions at pH 7 (lanes 1 and 2) by ultracentrifugation. Polypeptides were immunoprecipitated with anti-16-kDa protein serum, separated on an SDS-15% polyacrylamide gel, and detected by fluorography. Membrane (M) pellets were resuspended in hypotonic buffer; treated with 1% Triton X-100 (TX-100), 100 mM Na 2 CO 3 , or 1 M KCl; and further fractionated into supernatant (S) and pellet (P) fractions by ultracentrifugation. Polypeptides were immunoprecipitated with anti-16-kDa protein serum, separated on an SDS-15% polyacrylamide gel, and detected by fluorography.

    Journal: Journal of Virology

    Article Title: Membrane Association and Dimerization of a Cysteine-Rich, 16-Kilodalton Polypeptide Released from the C-Terminal Region of the Coronavirus Infectious Bronchitis Virus 1a Polyprotein

    doi: 10.1128/JVI.76.12.6257-6267.2002

    Figure Lengend Snippet: (a) Subcellular localization of the 16-kDa protein. (A) Cos-7 cells overexpressing pEGFP-16k at 16 h posttransfection showing perinuclear staining. (B) Cos-7 cells overexpressing the EGFP control vector (pEGFP) showing diffuse staining. (C) Immunofluorescent staining of IBV-infected Vero cells with anti-16-kDa protein serum (1:20) at 13 h postinfection showing distinct punctate perinuclear staining. (D) Immunofluorescent staining of mock-infected Vero cells with anti-16-kDa protein serum (1:20) at 13 h postinfection. (E) Double labeling of IBV-infected Vero cells that were transfected with BrUTP at 10 h postinfection showing the staining profile of anti-16-kDa protein serum. (F) Double labeling of IBV-infected Vero cells that were transfected with BrUTP at 10 h postinfection showing the staining pattern of anti-BrUTP serum. (G) Superimposition of images E and F. All images were taken from a Zeiss Axioplan confocal microscope. (b) Membrane association of the 16-kDa protein. Cos-7 cells expressing the 16-kDa protein were labeled with [ 35 S]methionine-cysteine for 4 h and harvested. Cells were lysed with a Dounce homogenizer and fractionated into membrane (M) and cytosol (C) fractions at pH 7 (lanes 1 and 2) by ultracentrifugation. Polypeptides were immunoprecipitated with anti-16-kDa protein serum, separated on an SDS-15% polyacrylamide gel, and detected by fluorography. Membrane (M) pellets were resuspended in hypotonic buffer; treated with 1% Triton X-100 (TX-100), 100 mM Na 2 CO 3 , or 1 M KCl; and further fractionated into supernatant (S) and pellet (P) fractions by ultracentrifugation. Polypeptides were immunoprecipitated with anti-16-kDa protein serum, separated on an SDS-15% polyacrylamide gel, and detected by fluorography.

    Article Snippet: To investigate this possibility, IBV-infected Vero cells were BrUTP labeled at 10 h postinfection for 4 h and double labeled with an anti-BrdU monoclonal antibody (Roche) and anti-16-kDa protein serum, showing perinuclear punctate vesicular staining patterns (Fig. , parts E and F).

    Techniques: Staining, Plasmid Preparation, Infection, Labeling, Transfection, Microscopy, Expressing, Immunoprecipitation

    (A) NF-Y is associated with active transcription sites in living cells. After in vivo incorporation of BrUTP (run-on), cells were fixed and endogenous NF-YA (ii) and nascent RNA transcripts (i) were detected by indirect immunofluorescence combined with Confocal Scanning Laser Microscopy by using anti-NF-YA and anti-BrU antibodies. In the overlay (iii), yellow indicates colocalizations between NF-YA (green) and transcription sites (red). In panels vi and x cells were immunostained with anti-NF-YA, in panels v and ix with anti-RPII CTD repeat YSPTSPS (phospho S2) and anti-total RPII respectively. The majority of NF-YA (red) colocalizes with the activated form of RPII (green)(vii). (B) Cells were immunostained with anti-NF-YA (vii-xii), -acetylated H3K9 (i), -acetylated H4 (ii), -tri-methylated H3K9 (iii), -di-methylated H3K27 (iv), -tri-methylated H4K20 (v) and -NF-YB (vi) antibodies. The majority of NF-YA colocalizes with acetylated (xiii, xiv), but poor colocalization occurs with methylated histones (xv, xvi,xvii). Panel xviii shows the overlay of two subunits of NF-Y, NF-YA (xii) and NF-YB (vi). Panels from xix to xxiv represent a typical optical field of the merge. In figure 1A and 1B confocal analysis of single optical section is shown. The images have been collected with a 60x objective.

    Journal: PLoS ONE

    Article Title: NF-Y Dependent Epigenetic Modifications Discriminate between Proliferating and Postmitotic Tissue

    doi: 10.1371/journal.pone.0002047

    Figure Lengend Snippet: (A) NF-Y is associated with active transcription sites in living cells. After in vivo incorporation of BrUTP (run-on), cells were fixed and endogenous NF-YA (ii) and nascent RNA transcripts (i) were detected by indirect immunofluorescence combined with Confocal Scanning Laser Microscopy by using anti-NF-YA and anti-BrU antibodies. In the overlay (iii), yellow indicates colocalizations between NF-YA (green) and transcription sites (red). In panels vi and x cells were immunostained with anti-NF-YA, in panels v and ix with anti-RPII CTD repeat YSPTSPS (phospho S2) and anti-total RPII respectively. The majority of NF-YA (red) colocalizes with the activated form of RPII (green)(vii). (B) Cells were immunostained with anti-NF-YA (vii-xii), -acetylated H3K9 (i), -acetylated H4 (ii), -tri-methylated H3K9 (iii), -di-methylated H3K27 (iv), -tri-methylated H4K20 (v) and -NF-YB (vi) antibodies. The majority of NF-YA colocalizes with acetylated (xiii, xiv), but poor colocalization occurs with methylated histones (xv, xvi,xvii). Panel xviii shows the overlay of two subunits of NF-Y, NF-YA (xii) and NF-YB (vi). Panels from xix to xxiv represent a typical optical field of the merge. In figure 1A and 1B confocal analysis of single optical section is shown. The images have been collected with a 60x objective.

    Article Snippet: The following primary antibodies (diluted in 1% BSA) were used: anti-NF-YA rabbit polyclonal (on run on experiments) (Rockland n°200-401-100, Gilbertsville, PA), anti-NF-YA mouse monoclonal from hybridoma cells, anti-NF-YB rabbit polyclonal (gifts from R. Mantovani), anti-BrUTP (Roche Diagnostics n°1170376, clone BMC 9318), anti-RPII (Santa Cruz n° 899), anti- RPIIS2 (Abcam n°5095), anti-PAN-H4ac (Upstate n°06-598), anti-H3ac-k9 (Abcam n°4441), anti-H3tri-m-k9 (Abcam n°8898), anti-H4tri-m-k20 (Abcam n°9053), anti-p300 (Santa Cruz n° 584 and 585), anti-H3di-m-k27 (Abcam n°24684) and anti-HDAC1 (Sigma H3284).

    Techniques: In Vivo, Immunofluorescence, Microscopy, Methylation