dsrna ladder  (New England Biolabs)


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    Name:
    dsRNA Ladder
    Description:
    dsRNA Ladder 25 gel lanes
    Catalog Number:
    n0363s
    Price:
    94
    Size:
    25 gel lanes
    Category:
    RNA Ladders
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    Structured Review

    New England Biolabs dsrna ladder
    dsRNA Ladder
    dsRNA Ladder 25 gel lanes
    https://www.bioz.com/result/dsrna ladder/product/New England Biolabs
    Average 94 stars, based on 61 article reviews
    Price from $9.99 to $1999.99
    dsrna ladder - by Bioz Stars, 2020-05
    94/100 stars

    Images

    1) Product Images from "TRIM25 binds RNA to modulate cellular anti-viral defense"

    Article Title: TRIM25 binds RNA to modulate cellular anti-viral defense

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2018.10.003

    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
    Figure Legend Snippet: RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Techniques Used: Activity Assay, In Vitro, Purification, Incubation

    2) Product Images from "Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein"

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00070

    Confocal imaging of dsRNA reporter N. benthamiana infected with TBSV, PVX, TCV, TRV, TuMV, and GFLV. Virus infection resulted in a variety of patterns of intracellular relocation of B2:GFP. Profound modifications of B2:GFP localization to large cytoplasmic aggregates were observed upon TBSV and PVX infections. Smaller cytoplasmic aggregates were observed upon TCV and TRV-infections and almost no modification in B2:GFP localization occurred upon TuMV and GFLV infection except for the near depletion of B2:GFP from the nucleoli (arrows, compare with Figure 4 ). Scale bars: 50 μm (upper panels) and 10 μm (lower panels).
    Figure Legend Snippet: Confocal imaging of dsRNA reporter N. benthamiana infected with TBSV, PVX, TCV, TRV, TuMV, and GFLV. Virus infection resulted in a variety of patterns of intracellular relocation of B2:GFP. Profound modifications of B2:GFP localization to large cytoplasmic aggregates were observed upon TBSV and PVX infections. Smaller cytoplasmic aggregates were observed upon TCV and TRV-infections and almost no modification in B2:GFP localization occurred upon TuMV and GFLV infection except for the near depletion of B2:GFP from the nucleoli (arrows, compare with Figure 4 ). Scale bars: 50 μm (upper panels) and 10 μm (lower panels).

    Techniques Used: Imaging, Infection, Modification

    Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) The dsRNA reporter remains associated with virus particles in the absence of an X-body during infection with PVX.ΔTGB1.GFP-CP (Tilsner et al., 2012 ). (C) dsRNA-containing granules between “whorls” of single-stranded vRNA labeled by Pumilio-BiFC (Tilsner et al., 2009 ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) as described for ssRNA (Tilsner et al., 2009 ). (H) Association and partial co-localization of the dsRNA reporter with peripheral membrane structures labeled by TGB3, which have been shown to be associated with plasmodesmata (Tilsner et al., 2013 ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.
    Figure Legend Snippet: Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) The dsRNA reporter remains associated with virus particles in the absence of an X-body during infection with PVX.ΔTGB1.GFP-CP (Tilsner et al., 2012 ). (C) dsRNA-containing granules between “whorls” of single-stranded vRNA labeled by Pumilio-BiFC (Tilsner et al., 2009 ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) as described for ssRNA (Tilsner et al., 2009 ). (H) Association and partial co-localization of the dsRNA reporter with peripheral membrane structures labeled by TGB3, which have been shown to be associated with plasmodesmata (Tilsner et al., 2013 ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.

    Techniques Used: Infection, Labeling, Bimolecular Fluorescence Complementation Assay, Blocking Assay, Marker

    Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.
    Figure Legend Snippet: Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.

    Techniques Used: Infection, Fluorescence, Marker

    Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.
    Figure Legend Snippet: Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.

    Techniques Used: Labeling, Infection

    Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).
    Figure Legend Snippet: Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).

    Techniques Used: Concentration Assay, Infection, Staining

    Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .
    Figure Legend Snippet: Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .

    Techniques Used: Fluorescence, Labeling, In Situ, Purification, Infection

    Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .
    Figure Legend Snippet: Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .

    Techniques Used: Imaging

    Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .
    Figure Legend Snippet: Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .

    Techniques Used: Imaging, Infection, Marker, Expressing, Labeling

    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).
    Figure Legend Snippet: Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Techniques Used: Binding Assay, In Vitro, Mobility Shift, Molecular Weight

    3) Product Images from "Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein"

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00070

    Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) ). (C) ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) ). (H) ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.
    Figure Legend Snippet: Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) ). (C) ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) ). (H) ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.

    Techniques Used: Infection, Blocking Assay, Marker, Labeling

    Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.
    Figure Legend Snippet: Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.

    Techniques Used: Infection, Fluorescence, Marker

    Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.
    Figure Legend Snippet: Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.

    Techniques Used: Labeling, Infection

    Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).
    Figure Legend Snippet: Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).

    Techniques Used: Concentration Assay, Infection, Staining

    Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .
    Figure Legend Snippet: Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .

    Techniques Used: Fluorescence, Labeling, In Situ, Purification, Infection

    Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .
    Figure Legend Snippet: Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .

    Techniques Used: Imaging

    Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .
    Figure Legend Snippet: Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .

    Techniques Used: Imaging, Infection, Marker, Expressing, Labeling

    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).
    Figure Legend Snippet: Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Techniques Used: Binding Assay, In Vitro, Mobility Shift, Molecular Weight

    4) Product Images from "Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: separation from chromosomal DNA by selective precipitation"

    Article Title: Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: separation from chromosomal DNA by selective precipitation

    Journal: Analytical biochemistry

    doi: 10.1016/j.ab.2015.09.017

    The lower and upper RNase-resistant bands are composed of RNA fragments ranging in size from 10-16 bp and 24-56 bp, respectively. (A) Lane 1, S. cerevisiae chromosomal DNA treated with RNase A was run on a 3.5% agarose gel; M, dsRNA ladder; (B) The logs
    Figure Legend Snippet: The lower and upper RNase-resistant bands are composed of RNA fragments ranging in size from 10-16 bp and 24-56 bp, respectively. (A) Lane 1, S. cerevisiae chromosomal DNA treated with RNase A was run on a 3.5% agarose gel; M, dsRNA ladder; (B) The logs

    Techniques Used: Agarose Gel Electrophoresis

    5) Product Images from "Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein"

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00070

    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).
    Figure Legend Snippet: Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Techniques Used: Binding Assay, In Vitro, Mobility Shift, Molecular Weight

    6) Product Images from "Detection of a Fourth Orbivirus Non-Structural Protein"

    Article Title: Detection of a Fourth Orbivirus Non-Structural Protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025697

    Colorimetric assay to detect interactions of NS4 with dsRNA. The graph shows colorimetric OD readings plotted against concentrations of dsRNA. Increasing concentrations (from 1 to 640 ng) of a biotinylated dsRNA were bound to wells coated with streptavidin. BTV NS4 or GIV NS4 were added to the wells in triplicate. Wells not containing dsRNA/NS4 were included as negative controls. Wells from which dsRNA was omitted, but in which NS4 (BTV or GIV) alone was incubated were also included as controls. Only wells containing the dsRNA to which GIV NS4 was added reacted with anti-GIV antibodies as indicated by increasing OD readings. The readings were almost linear (reaching a plateau at 320 ng of dsRNA) indicating that dsRNA acted as a target for binding of GIV NS4.
    Figure Legend Snippet: Colorimetric assay to detect interactions of NS4 with dsRNA. The graph shows colorimetric OD readings plotted against concentrations of dsRNA. Increasing concentrations (from 1 to 640 ng) of a biotinylated dsRNA were bound to wells coated with streptavidin. BTV NS4 or GIV NS4 were added to the wells in triplicate. Wells not containing dsRNA/NS4 were included as negative controls. Wells from which dsRNA was omitted, but in which NS4 (BTV or GIV) alone was incubated were also included as controls. Only wells containing the dsRNA to which GIV NS4 was added reacted with anti-GIV antibodies as indicated by increasing OD readings. The readings were almost linear (reaching a plateau at 320 ng of dsRNA) indicating that dsRNA acted as a target for binding of GIV NS4.

    Techniques Used: Colorimetric Assay, Incubation, Binding Assay

    Dicer competition assay. Lane RL: dsRNA ladder labelled in base pairs. Lane 1: dsRNA ladder pre-incubated with GIV NS4 followed by Dicer. GIV NS4 prevented cleavage by Dicer. Lane 2: dsRNA ladder pre-incubated with BTV-8 NS4, followed by Dicer. BTV-8 NS4 did not prevented Dicer from cleaving long dsRNAs into 21 bp long siRNAs. Lane 3: ladder incubated with Dicer as a positive digestion-control. Lane 4 : dsRNA ladder pre-incubated with VP9 of BAV followed by Dicer. VP9 of BAV did not prevent Dicer from cleaving long dsRNAs into 21 bp long siRNA. Lane 5: dsRNA ladder incubated with VP9 of BAV. VP9 of BAV did not affect the integrity of dsRNA.
    Figure Legend Snippet: Dicer competition assay. Lane RL: dsRNA ladder labelled in base pairs. Lane 1: dsRNA ladder pre-incubated with GIV NS4 followed by Dicer. GIV NS4 prevented cleavage by Dicer. Lane 2: dsRNA ladder pre-incubated with BTV-8 NS4, followed by Dicer. BTV-8 NS4 did not prevented Dicer from cleaving long dsRNAs into 21 bp long siRNAs. Lane 3: ladder incubated with Dicer as a positive digestion-control. Lane 4 : dsRNA ladder pre-incubated with VP9 of BAV followed by Dicer. VP9 of BAV did not prevent Dicer from cleaving long dsRNAs into 21 bp long siRNA. Lane 5: dsRNA ladder incubated with VP9 of BAV. VP9 of BAV did not affect the integrity of dsRNA.

    Techniques Used: Competitive Binding Assay, Incubation

    7) Product Images from "Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein"

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00070

    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).
    Figure Legend Snippet: Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Techniques Used: Binding Assay, In Vitro, Mobility Shift, Molecular Weight

    8) Product Images from "Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer"

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00787-16

    In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.
    Figure Legend Snippet: In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.

    Techniques Used: In Vitro, Activity Assay, Incubation, Labeling

    Related Articles

    Negative Control:

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
    Article Snippet: .. Reagents The following reagents were purchased: mouse mAb J2 (Scicons); Alexa Fluor 488-goat anti-mouse IgG secondary antibody (Molecular Probes, Life Technologies); Phi6 dsRNA (Finnzymes, Thermo Fisher Scientific); dsRNA ladder (New England Biolabs); High MW poly (A:U) and poly (I:C) (InvivoGen); GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); Silencer negative control 1 siRNA (Ambion, Life Technologies); Strep-Tactin conjugated to horseradish peroxidase (IBA Lifesciences); pNPP (Sigma-Aldrich), mMESSAGE mMACHINE T7 transcription kit (Ambion, Life Technologies), Lumi-Light western blotting substrate (Roche Diagnostics). ..

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
    Article Snippet: .. The following reagents were purchased: mouse mAb J2 (Scicons); Alexa Fluor 488-goat anti-mouse IgG secondary antibody (Molecular Probes, Life Technologies); Phi6 dsRNA (Finnzymes, Thermo Fisher Scientific); dsRNA ladder (New England Biolabs); High MW poly (A:U) and poly (I:C) (InvivoGen); GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); Silencer negative control 1 siRNA (Ambion, Life Technologies); Strep-Tactin conjugated to horseradish peroxidase (IBA Lifesciences); pNPP (Sigma-Aldrich), mMESSAGE mMACHINE T7 transcription kit (Ambion, Life Technologies), Lumi-Light western blotting substrate (Roche Diagnostics). ..

    Agarose Gel Electrophoresis:

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
    Article Snippet: .. Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol. .. Nucleic acids were visualized after ethidium bromide staining, using E-BOX VX5 gel imaging system (Vilber Lourmat), and fluorescent B2 protein was detected using G:BOX Chemi XRQ fluorescence imaging system (Syngene).

    Purification:

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
    Article Snippet: .. Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol. .. Nucleic acids were visualized after ethidium bromide staining, using E-BOX VX5 gel imaging system (Vilber Lourmat), and fluorescent B2 protein was detected using G:BOX Chemi XRQ fluorescence imaging system (Syngene).

    Incubation:

    Article Title: Detection of a Fourth Orbivirus Non-Structural Protein
    Article Snippet: .. Incubation of the dsRNA ladder with BTV NS4 or GIV NS4 alone did not alter dsRNA integrity. dsRNA preincubated with NS4 of GIV was protected against Dicer cleavage, consistent with previous findings regarding the presence of a dsRNA-binding domain. .. However, BTV NS4 did not protect dsRNA against Dicer and dsRNA was still processed into 21 bp long fragments, as analysed by agarose gel electrophoresis ( ).

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
    Article Snippet: .. Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol. .. Nucleic acids were visualized after ethidium bromide staining, using E-BOX VX5 gel imaging system (Vilber Lourmat), and fluorescent B2 protein was detected using G:BOX Chemi XRQ fluorescence imaging system (Syngene).

    other:

    Article Title: Rapid agarose gel electrophoretic mobility shift assay for quantitating protein:RNA interactions
    Article Snippet: Samples were as follows: 1, dsRNA ladder (New England BioLabs); 2, p19RNA-1; 3, p19RNA-2; 4, dsRNA stored at −20 °C; 5, freshly prepared dsRNA; 6, 48-nt RNA stem-loop; 7, 25mer ssRNA.

    Article Title: Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: separation from chromosomal DNA by selective precipitation
    Article Snippet: RNase If , 2-log DNA ladder, and dsRNA ladder were purchased from New England Biolabs.

    Western Blot:

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
    Article Snippet: .. Reagents The following reagents were purchased: mouse mAb J2 (Scicons); Alexa Fluor 488-goat anti-mouse IgG secondary antibody (Molecular Probes, Life Technologies); Phi6 dsRNA (Finnzymes, Thermo Fisher Scientific); dsRNA ladder (New England Biolabs); High MW poly (A:U) and poly (I:C) (InvivoGen); GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); Silencer negative control 1 siRNA (Ambion, Life Technologies); Strep-Tactin conjugated to horseradish peroxidase (IBA Lifesciences); pNPP (Sigma-Aldrich), mMESSAGE mMACHINE T7 transcription kit (Ambion, Life Technologies), Lumi-Light western blotting substrate (Roche Diagnostics). ..

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
    Article Snippet: .. The following reagents were purchased: mouse mAb J2 (Scicons); Alexa Fluor 488-goat anti-mouse IgG secondary antibody (Molecular Probes, Life Technologies); Phi6 dsRNA (Finnzymes, Thermo Fisher Scientific); dsRNA ladder (New England Biolabs); High MW poly (A:U) and poly (I:C) (InvivoGen); GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); Silencer negative control 1 siRNA (Ambion, Life Technologies); Strep-Tactin conjugated to horseradish peroxidase (IBA Lifesciences); pNPP (Sigma-Aldrich), mMESSAGE mMACHINE T7 transcription kit (Ambion, Life Technologies), Lumi-Light western blotting substrate (Roche Diagnostics). ..

    Binding Assay:

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
    Article Snippet: .. Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol. .. Nucleic acids were visualized after ethidium bromide staining, using E-BOX VX5 gel imaging system (Vilber Lourmat), and fluorescent B2 protein was detected using G:BOX Chemi XRQ fluorescence imaging system (Syngene).

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    New England Biolabs dsrna ladder
    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with <t>RNase</t> A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of <t>dsRNA</t> (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
    Dsrna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Journal: Journal of molecular biology

    Article Title: TRIM25 binds RNA to modulate cellular anti-viral defense

    doi: 10.1016/j.jmb.2018.10.003

    Figure Lengend Snippet: RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Article Snippet: Experiments in used RNase A (Qiagen), DNase I (Sigma), dsRNA ladder (NEB), and 100-bp dsDNA ladder (NEB).

    Techniques: Activity Assay, In Vitro, Purification, Incubation

    Confocal imaging of dsRNA reporter N. benthamiana infected with TBSV, PVX, TCV, TRV, TuMV, and GFLV. Virus infection resulted in a variety of patterns of intracellular relocation of B2:GFP. Profound modifications of B2:GFP localization to large cytoplasmic aggregates were observed upon TBSV and PVX infections. Smaller cytoplasmic aggregates were observed upon TCV and TRV-infections and almost no modification in B2:GFP localization occurred upon TuMV and GFLV infection except for the near depletion of B2:GFP from the nucleoli (arrows, compare with Figure 4 ). Scale bars: 50 μm (upper panels) and 10 μm (lower panels).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Confocal imaging of dsRNA reporter N. benthamiana infected with TBSV, PVX, TCV, TRV, TuMV, and GFLV. Virus infection resulted in a variety of patterns of intracellular relocation of B2:GFP. Profound modifications of B2:GFP localization to large cytoplasmic aggregates were observed upon TBSV and PVX infections. Smaller cytoplasmic aggregates were observed upon TCV and TRV-infections and almost no modification in B2:GFP localization occurred upon TuMV and GFLV infection except for the near depletion of B2:GFP from the nucleoli (arrows, compare with Figure 4 ). Scale bars: 50 μm (upper panels) and 10 μm (lower panels).

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Imaging, Infection, Modification

    Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) The dsRNA reporter remains associated with virus particles in the absence of an X-body during infection with PVX.ΔTGB1.GFP-CP (Tilsner et al., 2012 ). (C) dsRNA-containing granules between “whorls” of single-stranded vRNA labeled by Pumilio-BiFC (Tilsner et al., 2009 ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) as described for ssRNA (Tilsner et al., 2009 ). (H) Association and partial co-localization of the dsRNA reporter with peripheral membrane structures labeled by TGB3, which have been shown to be associated with plasmodesmata (Tilsner et al., 2013 ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) The dsRNA reporter remains associated with virus particles in the absence of an X-body during infection with PVX.ΔTGB1.GFP-CP (Tilsner et al., 2012 ). (C) dsRNA-containing granules between “whorls” of single-stranded vRNA labeled by Pumilio-BiFC (Tilsner et al., 2009 ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) as described for ssRNA (Tilsner et al., 2009 ). (H) Association and partial co-localization of the dsRNA reporter with peripheral membrane structures labeled by TGB3, which have been shown to be associated with plasmodesmata (Tilsner et al., 2013 ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Infection, Labeling, Bimolecular Fluorescence Complementation Assay, Blocking Assay, Marker

    Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Infection, Fluorescence, Marker

    Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Labeling, Infection

    Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Concentration Assay, Infection, Staining

    Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Fluorescence, Labeling, In Situ, Purification, Infection

    Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Imaging

    Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Imaging, Infection, Marker, Expressing, Labeling

    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Binding Assay, In Vitro, Mobility Shift, Molecular Weight

    Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) ). (C) ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) ). (H) ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) ). (C) ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) ). (H) ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Infection, Blocking Assay, Marker, Labeling

    Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Infection, Fluorescence, Marker

    Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Labeling, Infection

    Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Concentration Assay, Infection, Staining

    Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Fluorescence, Labeling, In Situ, Purification, Infection

    Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Imaging

    Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Imaging, Infection, Marker, Expressing, Labeling

    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Binding Assay, In Vitro, Mobility Shift, Molecular Weight

    The lower and upper RNase-resistant bands are composed of RNA fragments ranging in size from 10-16 bp and 24-56 bp, respectively. (A) Lane 1, S. cerevisiae chromosomal DNA treated with RNase A was run on a 3.5% agarose gel; M, dsRNA ladder; (B) The logs

    Journal: Analytical biochemistry

    Article Title: Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: separation from chromosomal DNA by selective precipitation

    doi: 10.1016/j.ab.2015.09.017

    Figure Lengend Snippet: The lower and upper RNase-resistant bands are composed of RNA fragments ranging in size from 10-16 bp and 24-56 bp, respectively. (A) Lane 1, S. cerevisiae chromosomal DNA treated with RNase A was run on a 3.5% agarose gel; M, dsRNA ladder; (B) The logs

    Article Snippet: RNase If , 2-log DNA ladder, and dsRNA ladder were purchased from New England Biolabs.

    Techniques: Agarose Gel Electrophoresis