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Colocalization of VDAC, ENO, and PYK in BaMV replication complex. ( A ) Schematic representation of the construction of an infectious clone for MS2-tagged BaMV, pKB sub MS2. Eight copies of bacteriophage MS2 coat protein binding sequence (8xMS2) were inserted into duplicated subgenomic promoter 2. The triangle represents sgp2. ( B ) Transient expression of MS2 FD -GFP, B2-OFP, and ENO-CFP with pKn or pKB sub MS2 in WT <t>N.</t> <t>benthamiana.</t> Images were taken at 3 dpa by confocal microscopy of agro-infiltrated leaves. CFP-, GFP-, and RFP, OFP-specific fluorescence is shown in blue, green, red, and orange, respectively. Chloroplast-derived autofluorescence is shown in violet. Z-projection images represent computer projections of multiple confocal sections (at least 20 sections per cell, each section approximately 0.5 μm focal depth). ( C ) BaMV VRC 3D image by Imaris image analysis software was created from pKBsubMS2-infected sample of B . The 3D model was vertically rotated 90 degree. Red represents B2-OFP signal. Violet surfaces were chloroplasts. Green represents BaMV viral RNA. ( D ) The size of BaMV VRC clusters of DMSO and <t>ENOblock</t> treated plants was calculated from Z-projection images. In DMSO and ENOblock treated plants, ten different BaMV-infected cells were selected and the size of BaMV VRC clusters was quantified and used for Student’s t -test. **** P < 0.0005. ( E ) BaMV level in DMSO- and ENOblock-treated plants was quantified by RT-qPCR from three plants, with the experiment repeated three times. Data from three independent experiments were analyzed using Student’s t -test. * P < 0.05. ( F ) Transient expression CFP-PYK, ENO-DsRed, and B2-GFP in mock- and BaMV-infected WT N. benthamiana at 3 dpa. Images of agro-infiltrated leaves were taken at 3 dpa by confocal microscopy. CFP-, GFP-, and RFP-specific fluorescence is shown in blue, green, and red or orange, respectively. The dashed circle indicates the nucleus. Chloroplast-derived autofluorescence is shown in violet. The triangle indicates B2-OFP signal closely associated with chloroplast. Z-projection images represent computer projections of multiple confocal sections (at least 20 sections per cell, each section approximately 0.5 μm focal depth).
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Colocalization of VDAC, ENO, and PYK in BaMV replication complex. ( A ) Schematic representation of the construction of an infectious clone for MS2-tagged BaMV, pKB sub MS2. Eight copies of bacteriophage MS2 coat protein binding sequence (8xMS2) were inserted into duplicated subgenomic promoter 2. The triangle represents sgp2. ( B ) Transient expression of MS2 FD -GFP, B2-OFP, and ENO-CFP with pKn or pKB sub MS2 in WT <t>N.</t> <t>benthamiana.</t> Images were taken at 3 dpa by confocal microscopy of agro-infiltrated leaves. CFP-, GFP-, and RFP, OFP-specific fluorescence is shown in blue, green, red, and orange, respectively. Chloroplast-derived autofluorescence is shown in violet. Z-projection images represent computer projections of multiple confocal sections (at least 20 sections per cell, each section approximately 0.5 μm focal depth). ( C ) BaMV VRC 3D image by Imaris image analysis software was created from pKBsubMS2-infected sample of B . The 3D model was vertically rotated 90 degree. Red represents B2-OFP signal. Violet surfaces were chloroplasts. Green represents BaMV viral RNA. ( D ) The size of BaMV VRC clusters of DMSO and <t>ENOblock</t> treated plants was calculated from Z-projection images. In DMSO and ENOblock treated plants, ten different BaMV-infected cells were selected and the size of BaMV VRC clusters was quantified and used for Student’s t -test. **** P < 0.0005. ( E ) BaMV level in DMSO- and ENOblock-treated plants was quantified by RT-qPCR from three plants, with the experiment repeated three times. Data from three independent experiments were analyzed using Student’s t -test. * P < 0.05. ( F ) Transient expression CFP-PYK, ENO-DsRed, and B2-GFP in mock- and BaMV-infected WT N. benthamiana at 3 dpa. Images of agro-infiltrated leaves were taken at 3 dpa by confocal microscopy. CFP-, GFP-, and RFP-specific fluorescence is shown in blue, green, and red or orange, respectively. The dashed circle indicates the nucleus. Chloroplast-derived autofluorescence is shown in violet. The triangle indicates B2-OFP signal closely associated with chloroplast. Z-projection images represent computer projections of multiple confocal sections (at least 20 sections per cell, each section approximately 0.5 μm focal depth).
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Colocalization of VDAC, ENO, and PYK in BaMV replication complex. ( A ) Schematic representation of the construction of an infectious clone for MS2-tagged BaMV, pKB sub MS2. Eight copies of bacteriophage MS2 coat protein binding sequence (8xMS2) were inserted into duplicated subgenomic promoter 2. The triangle represents sgp2. ( B ) Transient expression of MS2 FD -GFP, B2-OFP, and ENO-CFP with pKn or pKB sub MS2 in WT N. benthamiana. Images were taken at 3 dpa by confocal microscopy of agro-infiltrated leaves. CFP-, GFP-, and RFP, OFP-specific fluorescence is shown in blue, green, red, and orange, respectively. Chloroplast-derived autofluorescence is shown in violet. Z-projection images represent computer projections of multiple confocal sections (at least 20 sections per cell, each section approximately 0.5 μm focal depth). ( C ) BaMV VRC 3D image by Imaris image analysis software was created from pKBsubMS2-infected sample of B . The 3D model was vertically rotated 90 degree. Red represents B2-OFP signal. Violet surfaces were chloroplasts. Green represents BaMV viral RNA. ( D ) The size of BaMV VRC clusters of DMSO and ENOblock treated plants was calculated from Z-projection images. In DMSO and ENOblock treated plants, ten different BaMV-infected cells were selected and the size of BaMV VRC clusters was quantified and used for Student’s t -test. **** P < 0.0005. ( E ) BaMV level in DMSO- and ENOblock-treated plants was quantified by RT-qPCR from three plants, with the experiment repeated three times. Data from three independent experiments were analyzed using Student’s t -test. * P < 0.05. ( F ) Transient expression CFP-PYK, ENO-DsRed, and B2-GFP in mock- and BaMV-infected WT N. benthamiana at 3 dpa. Images of agro-infiltrated leaves were taken at 3 dpa by confocal microscopy. CFP-, GFP-, and RFP-specific fluorescence is shown in blue, green, and red or orange, respectively. The dashed circle indicates the nucleus. Chloroplast-derived autofluorescence is shown in violet. The triangle indicates B2-OFP signal closely associated with chloroplast. Z-projection images represent computer projections of multiple confocal sections (at least 20 sections per cell, each section approximately 0.5 μm focal depth).

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

Article Title: Proviral insights of glycolytic enolase in Bamboo mosaic virus replication associated with chloroplasts and mitochondria

doi: 10.1073/pnas.2415089122

Figure Lengend Snippet: Colocalization of VDAC, ENO, and PYK in BaMV replication complex. ( A ) Schematic representation of the construction of an infectious clone for MS2-tagged BaMV, pKB sub MS2. Eight copies of bacteriophage MS2 coat protein binding sequence (8xMS2) were inserted into duplicated subgenomic promoter 2. The triangle represents sgp2. ( B ) Transient expression of MS2 FD -GFP, B2-OFP, and ENO-CFP with pKn or pKB sub MS2 in WT N. benthamiana. Images were taken at 3 dpa by confocal microscopy of agro-infiltrated leaves. CFP-, GFP-, and RFP, OFP-specific fluorescence is shown in blue, green, red, and orange, respectively. Chloroplast-derived autofluorescence is shown in violet. Z-projection images represent computer projections of multiple confocal sections (at least 20 sections per cell, each section approximately 0.5 μm focal depth). ( C ) BaMV VRC 3D image by Imaris image analysis software was created from pKBsubMS2-infected sample of B . The 3D model was vertically rotated 90 degree. Red represents B2-OFP signal. Violet surfaces were chloroplasts. Green represents BaMV viral RNA. ( D ) The size of BaMV VRC clusters of DMSO and ENOblock treated plants was calculated from Z-projection images. In DMSO and ENOblock treated plants, ten different BaMV-infected cells were selected and the size of BaMV VRC clusters was quantified and used for Student’s t -test. **** P < 0.0005. ( E ) BaMV level in DMSO- and ENOblock-treated plants was quantified by RT-qPCR from three plants, with the experiment repeated three times. Data from three independent experiments were analyzed using Student’s t -test. * P < 0.05. ( F ) Transient expression CFP-PYK, ENO-DsRed, and B2-GFP in mock- and BaMV-infected WT N. benthamiana at 3 dpa. Images of agro-infiltrated leaves were taken at 3 dpa by confocal microscopy. CFP-, GFP-, and RFP-specific fluorescence is shown in blue, green, and red or orange, respectively. The dashed circle indicates the nucleus. Chloroplast-derived autofluorescence is shown in violet. The triangle indicates B2-OFP signal closely associated with chloroplast. Z-projection images represent computer projections of multiple confocal sections (at least 20 sections per cell, each section approximately 0.5 μm focal depth).

Article Snippet: Leaves of 3-wk-old WT N. benthamiana were infiltrated with DMSO or 20 μM ENOblock (MedChemExpress, Cat. No.: HY-15858) for 16 h and then agroinfiltrated with agrobacterium carrying pBin61-B2-GFP, pBin-ER-CFP, and pKn or pKB mixed in 1:1:1 ratio for 56 h. The area of cytosolic B2 signals embedded in ER was calculated from the Z-projection images.

Techniques: Protein Binding, Sequencing, Expressing, Confocal Microscopy, Fluorescence, Derivative Assay, Software, Infection, Quantitative RT-PCR