plasmid dna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher plasmid dna
    Role of Bax. ( a ) A-375 and A-375-TS were treated for 1 h with TRAIL (20 ng/ml)±BMS-345541 (A-375, 2 μ M; A-375-TS, 5 μ M) and were analyzed for Bax conformational changes by flow cytometry (Bax-NT). ( b ) Total protein extracts (caspase-8) and mitochondrial extracts (tBid and Bax) of A-375 and A-375-TS treated for 2 h were investigated by western blotting. Equal loading was proven by GAPDH and the mitochondrial protein VDAC, respectively. ( c ) A-375 and A-375-TS were treated for 1 h with BMS-345541 (2 μ M/5 μ M) and were analyzed by flow cytometry for total Bax, phosphorylated (p)Bax(Ser167) and pBax(Thr184) expression. Below, signals are shown for HCT-116 and HCT-116 Bax-KO cells. ( d ) A-375 and A-375-TS, treated with the same conditions, were analyzed after 24 h. ( e ) Protein expression (western blotting) of endogeneous I- κ B α (39 kDa) and I- κ B α -SR (45 kDa) is shown in mock- and I- κ B α <t>-SR-transfected</t> A-375 cells at 24 h. ( f ) Relative p65 <t>DNA-binding</t> capacity is shown in nuclear extracts of mock- and I- κ B α -SR-transfected A-375 cells, as quantified by enzyme-linked immunosorbent assay (ELISA) (24 h after transfection, 1 h treatment with TNF- α ). ( g ) Apoptosis and cytotoxicity were monitored in mock- or I- κ B α -SR-transfected A-375 treated in addition with BMS-345541 and/or TRAIL. Treatment with BMS-345541/TRAIL (for 24 h) started at 24 h after transfection. Mean values and S.D.'s of a representative experiment are shown (one of two, each with triplicates). ( h ) I- κ B α -SR- or mock-transfected A-375 cells were analyzed by flow cytometry at 24 h for Bax conformational changes (Bax-NT) and for Bax phosphorylation by pBax(Ser167) and pBax(Thr184) antibodies. For control, A-375 cells were treated in parallel for 24 h with BMS-345541 (2 μ M) and are shown below. For all flow cytometry data, treated cells are shown in overlays with respective non-treated controls and/or IgG1-stained controls. Always two independent experiments with triplicates revealed highly comparable results
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    Images

    1) Product Images from "Sensitization of melanoma cells for TRAIL-induced apoptosis by BMS-345541 correlates with altered phosphorylation and activation of Bax"

    Article Title: Sensitization of melanoma cells for TRAIL-induced apoptosis by BMS-345541 correlates with altered phosphorylation and activation of Bax

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.198

    Role of Bax. ( a ) A-375 and A-375-TS were treated for 1 h with TRAIL (20 ng/ml)±BMS-345541 (A-375, 2 μ M; A-375-TS, 5 μ M) and were analyzed for Bax conformational changes by flow cytometry (Bax-NT). ( b ) Total protein extracts (caspase-8) and mitochondrial extracts (tBid and Bax) of A-375 and A-375-TS treated for 2 h were investigated by western blotting. Equal loading was proven by GAPDH and the mitochondrial protein VDAC, respectively. ( c ) A-375 and A-375-TS were treated for 1 h with BMS-345541 (2 μ M/5 μ M) and were analyzed by flow cytometry for total Bax, phosphorylated (p)Bax(Ser167) and pBax(Thr184) expression. Below, signals are shown for HCT-116 and HCT-116 Bax-KO cells. ( d ) A-375 and A-375-TS, treated with the same conditions, were analyzed after 24 h. ( e ) Protein expression (western blotting) of endogeneous I- κ B α (39 kDa) and I- κ B α -SR (45 kDa) is shown in mock- and I- κ B α -SR-transfected A-375 cells at 24 h. ( f ) Relative p65 DNA-binding capacity is shown in nuclear extracts of mock- and I- κ B α -SR-transfected A-375 cells, as quantified by enzyme-linked immunosorbent assay (ELISA) (24 h after transfection, 1 h treatment with TNF- α ). ( g ) Apoptosis and cytotoxicity were monitored in mock- or I- κ B α -SR-transfected A-375 treated in addition with BMS-345541 and/or TRAIL. Treatment with BMS-345541/TRAIL (for 24 h) started at 24 h after transfection. Mean values and S.D.'s of a representative experiment are shown (one of two, each with triplicates). ( h ) I- κ B α -SR- or mock-transfected A-375 cells were analyzed by flow cytometry at 24 h for Bax conformational changes (Bax-NT) and for Bax phosphorylation by pBax(Ser167) and pBax(Thr184) antibodies. For control, A-375 cells were treated in parallel for 24 h with BMS-345541 (2 μ M) and are shown below. For all flow cytometry data, treated cells are shown in overlays with respective non-treated controls and/or IgG1-stained controls. Always two independent experiments with triplicates revealed highly comparable results
    Figure Legend Snippet: Role of Bax. ( a ) A-375 and A-375-TS were treated for 1 h with TRAIL (20 ng/ml)±BMS-345541 (A-375, 2 μ M; A-375-TS, 5 μ M) and were analyzed for Bax conformational changes by flow cytometry (Bax-NT). ( b ) Total protein extracts (caspase-8) and mitochondrial extracts (tBid and Bax) of A-375 and A-375-TS treated for 2 h were investigated by western blotting. Equal loading was proven by GAPDH and the mitochondrial protein VDAC, respectively. ( c ) A-375 and A-375-TS were treated for 1 h with BMS-345541 (2 μ M/5 μ M) and were analyzed by flow cytometry for total Bax, phosphorylated (p)Bax(Ser167) and pBax(Thr184) expression. Below, signals are shown for HCT-116 and HCT-116 Bax-KO cells. ( d ) A-375 and A-375-TS, treated with the same conditions, were analyzed after 24 h. ( e ) Protein expression (western blotting) of endogeneous I- κ B α (39 kDa) and I- κ B α -SR (45 kDa) is shown in mock- and I- κ B α -SR-transfected A-375 cells at 24 h. ( f ) Relative p65 DNA-binding capacity is shown in nuclear extracts of mock- and I- κ B α -SR-transfected A-375 cells, as quantified by enzyme-linked immunosorbent assay (ELISA) (24 h after transfection, 1 h treatment with TNF- α ). ( g ) Apoptosis and cytotoxicity were monitored in mock- or I- κ B α -SR-transfected A-375 treated in addition with BMS-345541 and/or TRAIL. Treatment with BMS-345541/TRAIL (for 24 h) started at 24 h after transfection. Mean values and S.D.'s of a representative experiment are shown (one of two, each with triplicates). ( h ) I- κ B α -SR- or mock-transfected A-375 cells were analyzed by flow cytometry at 24 h for Bax conformational changes (Bax-NT) and for Bax phosphorylation by pBax(Ser167) and pBax(Thr184) antibodies. For control, A-375 cells were treated in parallel for 24 h with BMS-345541 (2 μ M) and are shown below. For all flow cytometry data, treated cells are shown in overlays with respective non-treated controls and/or IgG1-stained controls. Always two independent experiments with triplicates revealed highly comparable results

    Techniques Used: Flow Cytometry, Cytometry, Western Blot, Expressing, Transfection, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining

    2) Product Images from "An RS Motif within the Epstein-Barr Virus BLRF2 Tegument Protein Is Phosphorylated by SRPK2 and Is Important for Viral Replication"

    Article Title: An RS Motif within the Epstein-Barr Virus BLRF2 Tegument Protein Is Phosphorylated by SRPK2 and Is Important for Viral Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053512

    BLRF2 rescues ORF52 null MHV68 replication, but the BLRF2-ARA mutant cannot. (A) Complementation assay measuring MHV68 virion release by quantitative PCR into supernatants of 293T cells co-transfected with replication defective MVH68 ORF52 null BAC and empty vector, wild-type FLAG-BLRF2, or FLAG-BLRF2-ARA mutant. Viral DNA was quantified four days post-transfection and the results shown are representative of two independent experiments performed in triplicate. Western blot with anti-flag antibody shows BLRF2-WT and –ARA expression levels.
    Figure Legend Snippet: BLRF2 rescues ORF52 null MHV68 replication, but the BLRF2-ARA mutant cannot. (A) Complementation assay measuring MHV68 virion release by quantitative PCR into supernatants of 293T cells co-transfected with replication defective MVH68 ORF52 null BAC and empty vector, wild-type FLAG-BLRF2, or FLAG-BLRF2-ARA mutant. Viral DNA was quantified four days post-transfection and the results shown are representative of two independent experiments performed in triplicate. Western blot with anti-flag antibody shows BLRF2-WT and –ARA expression levels.

    Techniques Used: Acetylene Reduction Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Transfection, BAC Assay, Plasmid Preparation, Western Blot, Expressing

    The BLRF2 ARA mutant exhibits increased cytoplasmic localization compared to wild-type BLRF2. (A) Immunofluorescence microcopy showing BLRF2 localization in HeLa cells transfected with BLRF2 wild-type or ARA mutant. Cells were stained with anti-BLRF2 antibody (green) and Draq5 DNA staining (blue). Examples of predominantly nuclear (left panels), mixed nuclear and cytoplamsic (middle panels), and predominantly cytoplasmic staining (right panels) are shown. (B) Summary of immunofluorescence analysis described in (A). The percentage of cells observed with predominantly nuclear (N) (blue), cytoplasmic (C) (red) or mixed (N and C) (gray) BLRF2 staining are shown.
    Figure Legend Snippet: The BLRF2 ARA mutant exhibits increased cytoplasmic localization compared to wild-type BLRF2. (A) Immunofluorescence microcopy showing BLRF2 localization in HeLa cells transfected with BLRF2 wild-type or ARA mutant. Cells were stained with anti-BLRF2 antibody (green) and Draq5 DNA staining (blue). Examples of predominantly nuclear (left panels), mixed nuclear and cytoplamsic (middle panels), and predominantly cytoplasmic staining (right panels) are shown. (B) Summary of immunofluorescence analysis described in (A). The percentage of cells observed with predominantly nuclear (N) (blue), cytoplasmic (C) (red) or mixed (N and C) (gray) BLRF2 staining are shown.

    Techniques Used: Acetylene Reduction Assay, Mutagenesis, Immunofluorescence, Transfection, Staining

    Characterization of BLRF2 during EBV replication. (A) Time course of EBV protein expression using whole cell lysates from P3HR1-ZHT cells (parental) or P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 induced with 4HT for 0, 24, 48 or 72 hours. Detection of tubulin serves as loading control. Endogenous BLRF2 is indicated with a solid arrowhead and FLAG-HA-BLRF2 with an open arrowhead. (B) Immunofluorescence microscopy to determine BLRF2 localization during EBV replication in P3HR1-ZHT cells (parental, top panel) or P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 (FLAG-HA-BLRF2, bottom panels), either uninduced (left) or induced with 4-Hydroxytamoxifen (4HT) for 48 hours (middle) or 72 hours (right). Anti-BLRF2 and anti-FLAG staining are shown in green and DNA staining is shown in blue. (C) Subcellular fractionation of EBV proteins and control cell proteins from P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 induced for replication with 4HT for 0, 24, 48 or 72 hours. Equal relative amounts of the cytosol (C), membrane and organelles (M), nucleus (N), chromatin bound (Ch) and cytoskeletal (Cs) fractions were probed for the indicated proteins. Tubulin served as a control for the cytosol fraction and Lamin B for the cytoskeleton fraction. As for panel A, endogenous and FLAG-HA tagged BLRF2 are indicated with filled and open arrowheads, respectively.
    Figure Legend Snippet: Characterization of BLRF2 during EBV replication. (A) Time course of EBV protein expression using whole cell lysates from P3HR1-ZHT cells (parental) or P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 induced with 4HT for 0, 24, 48 or 72 hours. Detection of tubulin serves as loading control. Endogenous BLRF2 is indicated with a solid arrowhead and FLAG-HA-BLRF2 with an open arrowhead. (B) Immunofluorescence microscopy to determine BLRF2 localization during EBV replication in P3HR1-ZHT cells (parental, top panel) or P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 (FLAG-HA-BLRF2, bottom panels), either uninduced (left) or induced with 4-Hydroxytamoxifen (4HT) for 48 hours (middle) or 72 hours (right). Anti-BLRF2 and anti-FLAG staining are shown in green and DNA staining is shown in blue. (C) Subcellular fractionation of EBV proteins and control cell proteins from P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 induced for replication with 4HT for 0, 24, 48 or 72 hours. Equal relative amounts of the cytosol (C), membrane and organelles (M), nucleus (N), chromatin bound (Ch) and cytoskeletal (Cs) fractions were probed for the indicated proteins. Tubulin served as a control for the cytosol fraction and Lamin B for the cytoskeleton fraction. As for panel A, endogenous and FLAG-HA tagged BLRF2 are indicated with filled and open arrowheads, respectively.

    Techniques Used: Expressing, Stable Transfection, Immunofluorescence, Microscopy, Staining, Fractionation

    3) Product Images from "Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells"

    Article Title: Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-8-45

    Gene expression profile in Rex1 KO ES cells . A . DNA microarray analysis of Rex1 KO ES cells. Scatter-plot of log-ratios of relative expression levels were shown for wild-type (EB5) versus Rex1 KO (HP3) ES cells. B . QPCR analyses of expressions of the putative Rex1 target genes. Three independent clones with each genotypes were cultured with LIF for 4 days, analyzed separately with normalization by the amount of Gapdh , and plotted with standard deviation against the expression level in undifferentiated wild-type ES cells (wt), set as 1.0.
    Figure Legend Snippet: Gene expression profile in Rex1 KO ES cells . A . DNA microarray analysis of Rex1 KO ES cells. Scatter-plot of log-ratios of relative expression levels were shown for wild-type (EB5) versus Rex1 KO (HP3) ES cells. B . QPCR analyses of expressions of the putative Rex1 target genes. Three independent clones with each genotypes were cultured with LIF for 4 days, analyzed separately with normalization by the amount of Gapdh , and plotted with standard deviation against the expression level in undifferentiated wild-type ES cells (wt), set as 1.0.

    Techniques Used: Expressing, Microarray, Real-time Polymerase Chain Reaction, Clone Assay, Cell Culture, Standard Deviation

    4) Product Images from "Generation of Functional Fluorescent BK Channels by Random Insertion of GFP Variants"

    Article Title: Generation of Functional Fluorescent BK Channels by Random Insertion of GFP Variants

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200509368

    55 fluorescent fusion hslo proteins were obtained with unique insertion sites. (A) AscI/EcoRI digestion was used to test the various hslo-YFP/CFP fusion constructs. Lane 1, 1 kb ladder DNA marker; lanes 2–6, representative hslo-YFP fusion proteins; lanes 7–11, representative hslo-CFP fusions. Note the common band (F) of 728 base pairs in every lane corresponding to CFP or YFP. The other two bands (H1, H2) show different sizes for each construct, depending on the site in which the transposon has been inserted. (B) Cell expression of fluorescent hslo-CFP or -YFP fusion proteins. Left panel, construct 829 (CFP); middle and right panels, constructs 1062 and 471, respectively (YFP). HEK293 cells were transiently transfected with the fusion constructs and imaged for fluorescence after 24 h. (C) Successful insertions of YFP or CFP into the different regions of the hslo α subunit. White circles, YFP; gray circles, CFP. Approximate insertion sites are shown. Refer to Table I for the exact position in the hslo sequence where the insertion occurred.
    Figure Legend Snippet: 55 fluorescent fusion hslo proteins were obtained with unique insertion sites. (A) AscI/EcoRI digestion was used to test the various hslo-YFP/CFP fusion constructs. Lane 1, 1 kb ladder DNA marker; lanes 2–6, representative hslo-YFP fusion proteins; lanes 7–11, representative hslo-CFP fusions. Note the common band (F) of 728 base pairs in every lane corresponding to CFP or YFP. The other two bands (H1, H2) show different sizes for each construct, depending on the site in which the transposon has been inserted. (B) Cell expression of fluorescent hslo-CFP or -YFP fusion proteins. Left panel, construct 829 (CFP); middle and right panels, constructs 1062 and 471, respectively (YFP). HEK293 cells were transiently transfected with the fusion constructs and imaged for fluorescence after 24 h. (C) Successful insertions of YFP or CFP into the different regions of the hslo α subunit. White circles, YFP; gray circles, CFP. Approximate insertion sites are shown. Refer to Table I for the exact position in the hslo sequence where the insertion occurred.

    Techniques Used: Construct, Marker, Expressing, Transfection, Fluorescence, Sequencing

    5) Product Images from "Silencing of miR-370 in Human Cholangiocarcinoma by Allelic Loss and Interleukin-6 Induced Maternal to Paternal Epigenotype Switch"

    Article Title: Silencing of miR-370 in Human Cholangiocarcinoma by Allelic Loss and Interleukin-6 Induced Maternal to Paternal Epigenotype Switch

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045606

    Methylation of IG-DMR-CG6 influences miR-370 expression. A. IG-DMR-CG6 is hypermethylated in human CCAs vs. matched normal tissues. Each line indicates a single clone, and each circle denotes the cytosine of a CpG site; filled and open circles represent methylated and unmethylated cytosines, respectively. B. miR-370 expression is inversely related to methylation at IG-DMR-CG6 in human liver specimens. X-axis - methylation rate, and miR-370 expression, Y-axis - human tissues. Percent methylation and miR-370 expression are expressed as a log2 of the ratio between the cancer and matched normal specimen. Since the data is expressed in logarithmic space, a positive value (as in the values for methylation for all 4 specimens) signifies more methylation in cancer vs. matched normal tissue. A negative value (as in the values for miR-370 for all 4 specimens) signifies a lower expression in cancer vs. matched normal specimen. These data demonstrates that for all 4 pairs of specimens, an increased in the methylation at IG-DMR-CG6 in cancer vs. matched normal is accompanied by a lower level of miR-370 in cancer vs. matched normal specimen. C. The expression of miR-370 is inversely correlated with level of DNA methylation. X-axis- miR-370 expression, Y-axis- overall methylation rate (%). r-correlation coefficient, p- P value.
    Figure Legend Snippet: Methylation of IG-DMR-CG6 influences miR-370 expression. A. IG-DMR-CG6 is hypermethylated in human CCAs vs. matched normal tissues. Each line indicates a single clone, and each circle denotes the cytosine of a CpG site; filled and open circles represent methylated and unmethylated cytosines, respectively. B. miR-370 expression is inversely related to methylation at IG-DMR-CG6 in human liver specimens. X-axis - methylation rate, and miR-370 expression, Y-axis - human tissues. Percent methylation and miR-370 expression are expressed as a log2 of the ratio between the cancer and matched normal specimen. Since the data is expressed in logarithmic space, a positive value (as in the values for methylation for all 4 specimens) signifies more methylation in cancer vs. matched normal tissue. A negative value (as in the values for miR-370 for all 4 specimens) signifies a lower expression in cancer vs. matched normal specimen. These data demonstrates that for all 4 pairs of specimens, an increased in the methylation at IG-DMR-CG6 in cancer vs. matched normal is accompanied by a lower level of miR-370 in cancer vs. matched normal specimen. C. The expression of miR-370 is inversely correlated with level of DNA methylation. X-axis- miR-370 expression, Y-axis- overall methylation rate (%). r-correlation coefficient, p- P value.

    Techniques Used: Methylation, Expressing, DNA Methylation Assay

    Two of the four CCA tissues displayed LOH at miR-370 locus vs. matched normal tissues. For each CCA specimen, we calculated the ratio between the level of miR-370 DNA in cancer vs. matched normal specimen. This ratio is displayed on the Y-axis. A ratio close to 1 (as shown for CCA1 and CCA2) signifies no LOH while a ratio close to 0.5 (as in CCA3 and CCA4) suggests LOH at miR-370 in CCA vs. matched normal tissue.
    Figure Legend Snippet: Two of the four CCA tissues displayed LOH at miR-370 locus vs. matched normal tissues. For each CCA specimen, we calculated the ratio between the level of miR-370 DNA in cancer vs. matched normal specimen. This ratio is displayed on the Y-axis. A ratio close to 1 (as shown for CCA1 and CCA2) signifies no LOH while a ratio close to 0.5 (as in CCA3 and CCA4) suggests LOH at miR-370 in CCA vs. matched normal tissue.

    Techniques Used:

    6) Product Images from "Variations in ribosomal DNA copy numbers in a genome of Trichophyton interdigitale. Variations in ribosomal DNA copy numbers in a genome of Trichophyton interdigitale"

    Article Title: Variations in ribosomal DNA copy numbers in a genome of Trichophyton interdigitale. Variations in ribosomal DNA copy numbers in a genome of Trichophyton interdigitale

    Journal: Mycoses

    doi: 10.1111/myc.13163

    Standard curve for cycle threshold (Ct) versus template DNA copies by qPCR. Serial dilutions of plasmid DNA containing the cloned internal transcribed spacer (ITS) region of rDNA (A) and TruMDR2 (MDR) (B) in Trichophyton interdigitale were tested by qPCR using sets of ITS and MDR primers. Each point represents the mean of triplicate measurements. The standard deviations of each point were too small to show in the graph
    Figure Legend Snippet: Standard curve for cycle threshold (Ct) versus template DNA copies by qPCR. Serial dilutions of plasmid DNA containing the cloned internal transcribed spacer (ITS) region of rDNA (A) and TruMDR2 (MDR) (B) in Trichophyton interdigitale were tested by qPCR using sets of ITS and MDR primers. Each point represents the mean of triplicate measurements. The standard deviations of each point were too small to show in the graph

    Techniques Used: Real-time Polymerase Chain Reaction, Plasmid Preparation, Clone Assay

    7) Product Images from "The integrity of chemically treated plasmid DNA as a chemical-based choice for prion clearance"

    Article Title: The integrity of chemically treated plasmid DNA as a chemical-based choice for prion clearance

    Journal: Regenerative Therapy

    doi: 10.1016/j.reth.2020.05.005

    GFP positive rate of HEK293 cells transfected with the plasmid DNA using Lipofectamine. Control is only precipitated by ethanol without chemically treatment. NC is only HEK293 cells, not transfected with plasmid DNA. (Leica Microsystems; magnification, 100 × ; scale bars, 100 μm). (A) The first DNA was treated with chemical reagents and precipitated by ethanol (Lot. B). (B) The second DNA was amplified and purified from the first DNA in E. coli (Lot. D). (C) The original DNA which was not amplified in E. coli and was purchased the plasmid DNA, transfected into HEK293 cells.
    Figure Legend Snippet: GFP positive rate of HEK293 cells transfected with the plasmid DNA using Lipofectamine. Control is only precipitated by ethanol without chemically treatment. NC is only HEK293 cells, not transfected with plasmid DNA. (Leica Microsystems; magnification, 100 × ; scale bars, 100 μm). (A) The first DNA was treated with chemical reagents and precipitated by ethanol (Lot. B). (B) The second DNA was amplified and purified from the first DNA in E. coli (Lot. D). (C) The original DNA which was not amplified in E. coli and was purchased the plasmid DNA, transfected into HEK293 cells.

    Techniques Used: Transfection, Plasmid Preparation, Amplification, Purification

    Transfection plasmid DNA into HEK293 cells. GFP protein transfecting plasmid DNA into HEK293 cells using Lipofectamine. The plasmid DNA was treated with chemical reagents and precipitated by ethanol (Leica Microsystems; magnification, 100 × ; scale bars, 100 μm). NC is only HEK293 cells, not transfected with plasmid DNA. (A) Transfection first plasmid DNA into HEK293 cells (Lot. B). (B) Transfection second plasmid DNA into HEK293 cells (Lot. D). (C) Transfection original plasmid DNA into HEK293 cells.
    Figure Legend Snippet: Transfection plasmid DNA into HEK293 cells. GFP protein transfecting plasmid DNA into HEK293 cells using Lipofectamine. The plasmid DNA was treated with chemical reagents and precipitated by ethanol (Leica Microsystems; magnification, 100 × ; scale bars, 100 μm). NC is only HEK293 cells, not transfected with plasmid DNA. (A) Transfection first plasmid DNA into HEK293 cells (Lot. B). (B) Transfection second plasmid DNA into HEK293 cells (Lot. D). (C) Transfection original plasmid DNA into HEK293 cells.

    Techniques Used: Transfection, Plasmid Preparation

    Integrity of DNA fragmentation using agarose gel electrophoresis and Bioanalyzer. The DNA fragmentation, pAcGFP1-C1 vector, has a length of 4731 bp. The ethanol precipitation was performed with a group of chemical reagents (Gdn-HCl, Gdn-SCN, TCA, or SDS) and in a group (Control) to which elution buffer was added instead of chemical reagents. The plasmid DNA without ethanol precipitation was named “Before.” The lower peak (aligned migration time, 35s) or upper peak (aligned migration time, 88s) of bioanalyzer’ electropherogram is marker. (A) Integrity of DNA fragmentation using agarose gel electrophoresis (Lot. C). (B) Integrity of chemically treated DNA fragmentation using Bioanalyzer (Lot. C). (C) Integrity of original DNA fragmentation using Bioanalyzer.
    Figure Legend Snippet: Integrity of DNA fragmentation using agarose gel electrophoresis and Bioanalyzer. The DNA fragmentation, pAcGFP1-C1 vector, has a length of 4731 bp. The ethanol precipitation was performed with a group of chemical reagents (Gdn-HCl, Gdn-SCN, TCA, or SDS) and in a group (Control) to which elution buffer was added instead of chemical reagents. The plasmid DNA without ethanol precipitation was named “Before.” The lower peak (aligned migration time, 35s) or upper peak (aligned migration time, 88s) of bioanalyzer’ electropherogram is marker. (A) Integrity of DNA fragmentation using agarose gel electrophoresis (Lot. C). (B) Integrity of chemically treated DNA fragmentation using Bioanalyzer (Lot. C). (C) Integrity of original DNA fragmentation using Bioanalyzer.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Ethanol Precipitation, Migration, Marker

    Related Articles

    Plasmid Preparation:

    Article Title: HPLC-DAD analysis, antioxidant potential and anti-urease activity of Asparagus gracilis collected from District Islamabad
    Article Snippet: .. Plasmid DNA (pBR322 Fermentas) 0.5 μg/3 μl was treated with 5 μl of each sample (100, 50 and 25 μg/ml). ..

    Article Title: Chemokine (C-C motif) ligand 2 (CCL2) mediates the pro-metastatic effect of dysadherin in human breast cancer cells
    Article Snippet: Quantification of CCL2 secretion in cell culture supernatants was performed by ELISA (Quantikine Immunoassay, R & D Systems), according to the manufacturer’s instructions. .. After knockdown or overexpression of dysadherin was performed as described above, cells in 12-well flat bottomed plates were transfected with a total of 250 ng/well of NF-κB-LUC promoter-reporter plasmid (a kind gift from Dr. Anita Roberts (NCI, Bethesda, MD)), together with 100 ng/well of pRL-TK ( Renilla ) plasmid DNA for normalization, using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. .. After transfection, cells were incubated for 16–18 hours, and cell lysates were harvested for assay using the Dual-Luciferase Reporter Assay System (Promega, Medison, WI).

    Article Title: Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation
    Article Snippet: In vivo transfection of p21WAF1/CIP1 .. p21 sense plasmid DNA Plasmid DNA was isolated from competent E-coli cells transformed with either the empty pcDNA3.1/Zeo vector (Invitrogen, Carlsbad CA) or the vector containing the full-length p21WAF1/CIP1 gene in the sense direction described by us [ ]. .. Mixed Lymphocyte Reaction (MLR) MLR was performed with splenocytes from isografts, untreated and CsA treated transplant recipients (LEW) as responders and irradiated splenocytes from donor strain (WF) as stimulators.

    Article Title: Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3
    Article Snippet: NF-κB-regulated luciferase activity was first normalized to Renilla luciferase activity, and then the fold activation relative to unstimulated conditions of each SseK3 variant was calculated. .. 4 × 105 293ET cells were transfected for 40 h with 1 μg of m6pPAC-FLAG-TRADD and 1 μg of m4pGFP-SseK3 variant (or 1 μg of m6pPAC-FLAG-GFP control) plasmid DNA using Lipofectamine 2000 (Invitrogen). .. Subsequently, the cells were harvested and lysed for 30 min on ice in lysis buffer (150 m m NaCl, 0.3% (v/v) Triton X-100, 20 m m Tris-Cl (pH 7.4), 5% glycerol, 5 m m EDTA, 1 m m phenylmethylsulfonyl fluoride, 1 m m benzamide, 2 μg/ml aprotinin, 5 μg/ml leupeptin, 1 m m DTT), and cytoplasmic proteins were isolated by a 30-min centrifugation at 16,000 × g at 4 °C.

    Article Title: Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea
    Article Snippet: The plates were then incubated at 37 °C for 16–18 h and the appearance of clear zones showing the antagonistic activity was observed. .. Isolation, purification and analysis of plasmid DNA Plasmid DNA from thirty one bacteriocinogenic S. sonnei was isolated using GeneJET Plasmid Miniprep Kit (Fermentas). .. DNA was visualized following electrophoresis in 0.7% (w/v) agarose gel in TBE buffer by staining with ethidium bromide (0.5 μg/mL).

    Article Title: Antioxidant Potential, DNA Protection, and HPLC-DAD Analysis of Neglected Medicinal Jurinea dolomiaea Roots
    Article Snippet: .. Plasmid DNA (pBR322 Fermentas) 0.5 μ g/3 μ L was treated with 5 μ L of each sample (100, 50, and 25 μ g/mL). ..

    Article Title: Splicing variation of Long-IRBIT determines the target selectivity of IRBIT family proteins
    Article Snippet: Gastric glands were pipetted onto the glass-bottom dish coated with Matrigel (BD). .. After 1 ∼2 h, transfection was performed using TransIT-LT1 Transfection Reagent (Mirus) according to the manufacturer’s instructions [plasmid DNA 3 μg, TransIT-LT1 12 μL, optiMEM (ThermoFisher) 250 μL for 3.5-cm glass-bottom dish]. .. Transfection efficiency for mouse gastric glands was ∼10%.

    Article Title: Scientific Validation of Ethnomedicinal Use of Ipomoea batatas L. Lam. as Aphrodisiac and Gonadoprotective Agent against Bisphenol A Induced Testicular Toxicity in Male Sprague Dawley Rats
    Article Snippet: .. According to protocol 0.5 μ g/3 μ l of Plasmid DNA (pBR322 Fermentas) was treated with 5 μ l of sample (100 μ g/ml). ..

    Over Expression:

    Article Title: Chemokine (C-C motif) ligand 2 (CCL2) mediates the pro-metastatic effect of dysadherin in human breast cancer cells
    Article Snippet: Quantification of CCL2 secretion in cell culture supernatants was performed by ELISA (Quantikine Immunoassay, R & D Systems), according to the manufacturer’s instructions. .. After knockdown or overexpression of dysadherin was performed as described above, cells in 12-well flat bottomed plates were transfected with a total of 250 ng/well of NF-κB-LUC promoter-reporter plasmid (a kind gift from Dr. Anita Roberts (NCI, Bethesda, MD)), together with 100 ng/well of pRL-TK ( Renilla ) plasmid DNA for normalization, using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. .. After transfection, cells were incubated for 16–18 hours, and cell lysates were harvested for assay using the Dual-Luciferase Reporter Assay System (Promega, Medison, WI).

    Transfection:

    Article Title: Chemokine (C-C motif) ligand 2 (CCL2) mediates the pro-metastatic effect of dysadherin in human breast cancer cells
    Article Snippet: Quantification of CCL2 secretion in cell culture supernatants was performed by ELISA (Quantikine Immunoassay, R & D Systems), according to the manufacturer’s instructions. .. After knockdown or overexpression of dysadherin was performed as described above, cells in 12-well flat bottomed plates were transfected with a total of 250 ng/well of NF-κB-LUC promoter-reporter plasmid (a kind gift from Dr. Anita Roberts (NCI, Bethesda, MD)), together with 100 ng/well of pRL-TK ( Renilla ) plasmid DNA for normalization, using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. .. After transfection, cells were incubated for 16–18 hours, and cell lysates were harvested for assay using the Dual-Luciferase Reporter Assay System (Promega, Medison, WI).

    Article Title: Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3
    Article Snippet: NF-κB-regulated luciferase activity was first normalized to Renilla luciferase activity, and then the fold activation relative to unstimulated conditions of each SseK3 variant was calculated. .. 4 × 105 293ET cells were transfected for 40 h with 1 μg of m6pPAC-FLAG-TRADD and 1 μg of m4pGFP-SseK3 variant (or 1 μg of m6pPAC-FLAG-GFP control) plasmid DNA using Lipofectamine 2000 (Invitrogen). .. Subsequently, the cells were harvested and lysed for 30 min on ice in lysis buffer (150 m m NaCl, 0.3% (v/v) Triton X-100, 20 m m Tris-Cl (pH 7.4), 5% glycerol, 5 m m EDTA, 1 m m phenylmethylsulfonyl fluoride, 1 m m benzamide, 2 μg/ml aprotinin, 5 μg/ml leupeptin, 1 m m DTT), and cytoplasmic proteins were isolated by a 30-min centrifugation at 16,000 × g at 4 °C.

    Article Title: Splicing variation of Long-IRBIT determines the target selectivity of IRBIT family proteins
    Article Snippet: Gastric glands were pipetted onto the glass-bottom dish coated with Matrigel (BD). .. After 1 ∼2 h, transfection was performed using TransIT-LT1 Transfection Reagent (Mirus) according to the manufacturer’s instructions [plasmid DNA 3 μg, TransIT-LT1 12 μL, optiMEM (ThermoFisher) 250 μL for 3.5-cm glass-bottom dish]. .. Transfection efficiency for mouse gastric glands was ∼10%.

    Isolation:

    Article Title: Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation
    Article Snippet: In vivo transfection of p21WAF1/CIP1 .. p21 sense plasmid DNA Plasmid DNA was isolated from competent E-coli cells transformed with either the empty pcDNA3.1/Zeo vector (Invitrogen, Carlsbad CA) or the vector containing the full-length p21WAF1/CIP1 gene in the sense direction described by us [ ]. .. Mixed Lymphocyte Reaction (MLR) MLR was performed with splenocytes from isografts, untreated and CsA treated transplant recipients (LEW) as responders and irradiated splenocytes from donor strain (WF) as stimulators.

    Article Title: Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea
    Article Snippet: The plates were then incubated at 37 °C for 16–18 h and the appearance of clear zones showing the antagonistic activity was observed. .. Isolation, purification and analysis of plasmid DNA Plasmid DNA from thirty one bacteriocinogenic S. sonnei was isolated using GeneJET Plasmid Miniprep Kit (Fermentas). .. DNA was visualized following electrophoresis in 0.7% (w/v) agarose gel in TBE buffer by staining with ethidium bromide (0.5 μg/mL).

    Transformation Assay:

    Article Title: Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation
    Article Snippet: In vivo transfection of p21WAF1/CIP1 .. p21 sense plasmid DNA Plasmid DNA was isolated from competent E-coli cells transformed with either the empty pcDNA3.1/Zeo vector (Invitrogen, Carlsbad CA) or the vector containing the full-length p21WAF1/CIP1 gene in the sense direction described by us [ ]. .. Mixed Lymphocyte Reaction (MLR) MLR was performed with splenocytes from isografts, untreated and CsA treated transplant recipients (LEW) as responders and irradiated splenocytes from donor strain (WF) as stimulators.

    Variant Assay:

    Article Title: Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3
    Article Snippet: NF-κB-regulated luciferase activity was first normalized to Renilla luciferase activity, and then the fold activation relative to unstimulated conditions of each SseK3 variant was calculated. .. 4 × 105 293ET cells were transfected for 40 h with 1 μg of m6pPAC-FLAG-TRADD and 1 μg of m4pGFP-SseK3 variant (or 1 μg of m6pPAC-FLAG-GFP control) plasmid DNA using Lipofectamine 2000 (Invitrogen). .. Subsequently, the cells were harvested and lysed for 30 min on ice in lysis buffer (150 m m NaCl, 0.3% (v/v) Triton X-100, 20 m m Tris-Cl (pH 7.4), 5% glycerol, 5 m m EDTA, 1 m m phenylmethylsulfonyl fluoride, 1 m m benzamide, 2 μg/ml aprotinin, 5 μg/ml leupeptin, 1 m m DTT), and cytoplasmic proteins were isolated by a 30-min centrifugation at 16,000 × g at 4 °C.

    Purification:

    Article Title: Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea
    Article Snippet: The plates were then incubated at 37 °C for 16–18 h and the appearance of clear zones showing the antagonistic activity was observed. .. Isolation, purification and analysis of plasmid DNA Plasmid DNA from thirty one bacteriocinogenic S. sonnei was isolated using GeneJET Plasmid Miniprep Kit (Fermentas). .. DNA was visualized following electrophoresis in 0.7% (w/v) agarose gel in TBE buffer by staining with ethidium bromide (0.5 μg/mL).

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  • 86
    Thermo Fisher plasmid dna
    Standard curves of <t>Saccharibacteria</t> qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid <t>DNA</t> carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.
    Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher dna plasmids
    Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were <t>transfected</t> by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB <t>DNA-binding</t> complexes using a 32 P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.
    Dna Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher plasmid dna harboring microcystis its sequences
    Phylogenetic tree for the six <t>Microcystis</t> genotypes generated based on the internal transcribed spacer <t>(ITS)</t> region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.
    Plasmid Dna Harboring Microcystis Its Sequences, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Journal: Materials

    Article Title: Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge

    doi: 10.3390/ma11071129

    Figure Lengend Snippet: Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Article Snippet: Standard curves were constructed from 10-fold dilutions of plasmid DNA (pCR2.1-TOPO, Thermo Fisher Scientific) carrying partial Saccharibacteria 16S rRNA genes retrieved from a clone library described in section 3.4 with a known copy number.

    Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification

    Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were transfected by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB DNA-binding complexes using a 32 P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.

    Journal: Journal of Virology

    Article Title: Role of NF-?B and Myc Proteins in Apoptosis Induced by Hepatitis B Virus HBx Protein

    doi: 10.1128/JVI.75.1.215-225.2001

    Figure Lengend Snippet: Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were transfected by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB DNA-binding complexes using a 32 P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.

    Article Snippet: Cells at 50% confluency were transfected with DNA plasmids using Lipofectin (Gibco) with a total of 10 μg of plasmid DNA per 10-cm plate of cells.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Transduction, Binding Assay, Labeling, Exclusion Assay

    Phylogenetic tree for the six Microcystis genotypes generated based on the internal transcribed spacer (ITS) region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.

    Journal: PLoS ONE

    Article Title: Biodiversity of cyanobacteria and other aquatic microorganisms across a freshwater to brackish water gradient determined by shotgun metagenomic sequencing analysis in the San Francisco Estuary, USA

    doi: 10.1371/journal.pone.0203953

    Figure Lengend Snippet: Phylogenetic tree for the six Microcystis genotypes generated based on the internal transcribed spacer (ITS) region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.

    Article Snippet: Standard curves were generated by running reactions on serial dilutions of plasmid DNA harboring Microcystis ITS sequences, which were chemically synthesized and ligated into a cloning vector by a manufacturer (ThermoFisher Scientific).

    Techniques: Generated, Sequencing

    Dynamitin overexpression inhibits nuclear targeting of incoming Ad2, but not release of a fluid-phase endosomal marker into the cytosol. (A) TR-labeled wt virus was bound in the cold to HeLa cells transiently transfected with a plasmid DNA expressing eGFP (panels a–c, arrows) or with the eGFP-plasmid together with a dynamitin/p50 (Dyn)-expressing plasmid (panels d–f). In the latter case, 95% of the GFP expressing cells also overexpressed dynamitin. Virus was internalized for 60 min at 37°C and cells were processed for indirect immunofluorescence microscopy using anti-dynamitin mAb 50.1 and goat anti–mouse IgG-Cy5. (B) wt Ad2 (panels a–c) or ts1 (panels d–f) was bound at 50 μg/ml to the surface of HeLa cells, which had been transiently transfected with dynamitin/p50 DNA. Virus was internalized for 15 min in the presence of FITC-labeled 10 K dextran (5 mg/ml), followed by a 15-min incubation in the absence of dextran, fixed with pFA, and then stained for dynamitin using mAb 50.1 and anti-mouse IgG-Cy5 (panels b and e). The distribution of dextran was recorded in the FITC channel (panels a and d, transfected cells are indicated by an arrow) and DIC images of the same cells shown in panels c and f.

    Journal: The Journal of Cell Biology

    Article Title: Microtubule-dependent Plus- and Minus End-directed Motilities Are Competing Processes for Nuclear Targeting of Adenovirus

    doi:

    Figure Lengend Snippet: Dynamitin overexpression inhibits nuclear targeting of incoming Ad2, but not release of a fluid-phase endosomal marker into the cytosol. (A) TR-labeled wt virus was bound in the cold to HeLa cells transiently transfected with a plasmid DNA expressing eGFP (panels a–c, arrows) or with the eGFP-plasmid together with a dynamitin/p50 (Dyn)-expressing plasmid (panels d–f). In the latter case, 95% of the GFP expressing cells also overexpressed dynamitin. Virus was internalized for 60 min at 37°C and cells were processed for indirect immunofluorescence microscopy using anti-dynamitin mAb 50.1 and goat anti–mouse IgG-Cy5. (B) wt Ad2 (panels a–c) or ts1 (panels d–f) was bound at 50 μg/ml to the surface of HeLa cells, which had been transiently transfected with dynamitin/p50 DNA. Virus was internalized for 15 min in the presence of FITC-labeled 10 K dextran (5 mg/ml), followed by a 15-min incubation in the absence of dextran, fixed with pFA, and then stained for dynamitin using mAb 50.1 and anti-mouse IgG-Cy5 (panels b and e). The distribution of dextran was recorded in the FITC channel (panels a and d, transfected cells are indicated by an arrow) and DIC images of the same cells shown in panels c and f.

    Article Snippet: Plasmids and DNA Experiments GFP-encoding plasmid DNA (gift of S. Zimmermann; Invitrogen, Zürich, Switzerland) was transfected into TC7 or HeLa cells alone or together with dynamitin-encoding DNA (gift of R. Vallee, University of Massachusetts, Worcester, MA) ( ) at a molar ratio of 2:1 using Lipofectamine™ (5 μl/μg DNA; GIBCO BRL ) or TransFast™ (7 μl/μg DNA; Promega ).

    Techniques: Over Expression, Marker, Labeling, Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Microscopy, Incubation, Staining