brutp  (Roche)

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

Journal:

Article Title: RNA-Templated Replication of Hepatitis Delta Virus: Genomic and Antigenomic RNAs Associate with Different Nuclear Bodies

doi: 10.1128/JVI.02650-05

Figure Lengend Snippet: Subcellular distribution of HDV RNA synthesis. BrUTP was incorporated (15-min labeling) into newly synthesized RNA and visualized by immunostaining (Cy2, green staining). (A) BrUTP incorporation in either the stable HDV cDNA-integrated cell line H1δ9

Article Snippet: For BrUTP and HDV RNA cotransfection, DOTAP (Roche) was used.

Techniques: Labeling, Synthesized, Immunostaining, Staining

Journal:

Article Title: RNA-Templated Replication of Hepatitis Delta Virus: Genomic and Antigenomic RNAs Associate with Different Nuclear Bodies

doi: 10.1128/JVI.02650-05

Figure Lengend Snippet: (A) HDV genomic RNA synthesis is colocalized with the PML body. The BrUTP-labeled RNA and PML body are identified by red (Cy3) and green (FITC) staining, respectively. The arrows indicate the convergence of PML body and BrUTP labeling, and the asterisks

Article Snippet: For BrUTP and HDV RNA cotransfection, DOTAP (Roche) was used.

Techniques: Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A-kinase-interacting protein localizes protein kinase A in the nucleus

doi: 10.1073/pnas.0408608102

Figure Lengend Snippet: The C subunit, TFIID, and BrUTP partially overlap with AKIP1. Hs 578 Bst cells were double-labeled with anti-AKIP1 antibody and BrdUrd, TFIID, C subunit, or PKI antibodies and visualized with confocal microscopy. The C subunit shows partial colocalization with AKIP1. There was partial overlap of AKIP1 with TFIID and BrUTP. Although PKI is present as distinct punctuate spots, it does not appear to overlap with AKIP1.

Article Snippet: The BrUTP incorporation assay was performed by using Hs 578 Bst as described with slight modifications ( ) and by Roche Applied Biosciences.

Techniques: Labeling, Confocal Microscopy

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