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  • 99
    Thermo Fisher xcell surelock mini cell system
    Xcell Surelock Mini Cell System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore acrylamide bisacrylamide
    Acrylamide Bisacrylamide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gel electrophoresis set up
    Gel Electrophoresis Set Up, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad pre stained sds page standards
    Pre Stained Sds Page Standards, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nitrocellulose membranes
    Nitrocellulose Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore polyvinylidene difluoride pvdf membrane
    Polyvinylidene Difluoride Pvdf Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ohara Inc ohara s lah79 n
    Ohara S Lah79 N, supplied by Ohara Inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad mini protean tbe gel
    Mini Protean Tbe Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hek293 cells
    Setting up the xCELLigence RTCA program schedule–Steps and Sweeps. An example of a Schedule page is shown for <t>HEK293</t> cells. Step 1 (A) is preprogrammed and should not be changed. Use Add a Step and/or Add a Substep (A) to insert additional experimental steps with different sweeps and intervals in the schedule (B).
    Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 11982 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad dna binding pcr plate
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Dna Binding Pcr Plate, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher xcell iitm blot module
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Xcell Iitm Blot Module, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lambda dna standard
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Lambda Dna Standard, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad horizontal electrophoresis chamber
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Horizontal Electrophoresis Chamber, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher power sybr green pcr master mix
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Power Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53341 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lightshift chemiluminescent emsa kit
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Lightshift Chemiluminescent Emsa Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lightshift chemiluminescent rna emsa kit
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Lightshift Chemiluminescent Rna Emsa Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gradient bis tris nupage gels
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Gradient Bis Tris Nupage Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reaction buffer
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5752 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad powerpac
    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed <t>PCR.</t> A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid <t>DNA</t> content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.
    Powerpac, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hyperimmune horse anti cvb3 serum
    ) or <t>CVB3-0</t> (0) or cell culture medium (control). CVB3-binding isotype was detected by ELISA using peroxidase activity of bound goat anti-mouse IgG1 or bound goat anti-mouse IgG2a and quantitated by absorbance at 405 nm.
    Hyperimmune Horse Anti Cvb3 Serum, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore low melting point agarose
    ) or <t>CVB3-0</t> (0) or cell culture medium (control). CVB3-binding isotype was detected by ELISA using peroxidase activity of bound goat anti-mouse IgG1 or bound goat anti-mouse IgG2a and quantitated by absorbance at 405 nm.
    Low Melting Point Agarose, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa premix taq
    ) or <t>CVB3-0</t> (0) or cell culture medium (control). CVB3-binding isotype was detected by ELISA using peroxidase activity of bound goat anti-mouse IgG1 or bound goat anti-mouse IgG2a and quantitated by absorbance at 405 nm.
    Premix Taq, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1473 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology goat β actin antibody
    The expression of RAD51 allele from various pYNRAD51 plasmids. ( A ) RT–PCR analysis of total RNA extracts from LSY678(Int) strain harboring various pYNRAD51 mutant plasmids. A 1.2 kb RAD51 product was amplified using primers of Rad51F and Rad51B and electrophoresed through 1% agarose gel. ( B ) Western blot analysis of whole-cell extracts from LSY402(Δrad51) and LSY678(Int) containing the various pYNRAD51 expression plasmids. Protein extracts from LSY402(Δrad51) or LSY678(Int), alone or containing the different pYNRAD51 expression plasmids, were evaluated by blotting with rabbit α Rad51 antibody and visualized by an anti-rabbit antibody conjugated with HRP in an enhanced chemiluminescence ECL™ system. Protein extracts from LSY402(Δrad51) with empty vector (pYN132) were used as a negative control. An internal control was set up by utilizing a goat <t>β-actin</t> antibody and visualized by anti-goat IgG-HRP antibody in the same ECL™ system.
    Goat β Actin Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam lactoferrin
    Mass spectrometry indicates that human CM <t>lactoferrin</t> binds DC-SIGN. (A) 4–12% SDS PAGE gel loaded (from left to right) with a 250kD protein marker, agarose beads, DC-SIGN-Fc, the supernatant from the first wash, supernatant from the second wash, and the DC-SIGN-Fc coated agarose beads loaded with the DC-SIGN binding component from CM. Band #2, 3 and 4 potentially contain the DC-SIGN binding component of CM. Ion trap mass spectrometry of in gel digests identified human lactoferrin fragments (highly abundant in band #3) and immunoglobulins in all three bands and intelectin-1 in band #4 (with trace amounts in the other bands). (B) CM representing a high DC-SIGN binder, an intermediate DC-SIGN binder and a low/no DC-SIGN binder were coated on an ELISA plate and were tested for DC-SIGN and lactoferrin binding. The first graph (left) confirms the DC-SIGN binding status while the second graph (right) shows the binding capacity of polyclonal anti-lactoferrin, which is high for CM from a DC-SIGN high binder, intermediate for an intermediate DC-SIGN binder and not present in CM from a low/no DC-SIGN binder.
    Lactoferrin, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega micrococcal nuclease treated rabbit reticulocyte lysate
    IRES-mediated translation and IRES inhibition in a cell-free system and in cells. (A) Diagrammatic comparison of general protein synthesis to IRES-mediated translation. General protein synthesis is mediated by cap-dependent ribosomal scanning from the 5′-end of the mRNA and may be modulated by mTOR inhibitors. Internal ribosome entry sites (IRESs) allow the 40S ribosome to engage the mRNA at a position much closer (in many cases immediately adjacent to) the AUG initiation codon. IRES-mediated translation is independently regulated and serves as a fail-safe mechanism ensuring the synthesis of proteins most critical for cell survival. (B) Structure of IRES inhibitor lead compound W (cpd_W): Ethyl 2-{[2-(1,3-benzoxazol-2-ylthio)butanoyl]amino}-4-methyl-1,3-thiazole-5-carboxylate, MW 405. (C) In vitro translation assays: <t>Rabbit</t> <t>reticulocyte</t> <t>lysate</t> was programmed with a bicistronic reporter RNA in which translation of the second cistron (firefly luciferase coding sequence) is mediated by the IGF1R IRES, while translation of the first cistron ( Renilla luciferase coding sequence) is mediated by ribosomal scanning. IRES inhibitor cpd_W (or vehicle control) was included in the reaction in increasing concentrations as indicated. The result is indicative of selective inhibition of IRES-mediated translation. A structural analog of cpd_W (W-7) in which a single atom has been modified (converting the benzoxazole to a benzimidazole) was completely inactive in this assay, indicative of the chemical specificity of IRES inhibition. Cycloheximide (5 µg/ml, chx) and puromycin (250 µg/ml, puro) were included as reference standards for non-specific translational inhibition (far right). (D) IRES inhibitor cpd_W completely blocked de novo synthesis of IGF1R in breast tumor cells under adverse conditions (serum-deprivation, loss of adhesion) relevant to the microenvironment of the tumor. T47D breast tumor cells were seeded in 6-well plates and allowed 48 h to recover and resume proliferation, then incubated in the presence of IRES inhibitor cpd_W (10 µg/ml) or vehicle control (0.1% DMSO) as indicated. The cells were simultaneously subjected to acute serum deprivation (0.5% fetal calf serum, no added insulin) to increase dependence on IRES-mediated translation. After 24 h, the cells were harvested and whole cell lysates prepared, equivalent aliquots separated by SDS-PAGE and immunoblotted for IGF1R-β and α-tubulin. In lanes 7–12, the cells were trypsinized and seeded into 6-well plates and immediately incubated in the presence of IRES-inhibitor cpd_W or vehicle control as indicated. Robust regeneration of trypsin-catabolized IGF1R was observed within 24 h in <t>vehicle-treated</t> cells, however, this was completely blocked in the presence of cpd_W (10 µg/ml as shown; IC 50 , 2 µg/ml). The asterisk (*) marks the position of trypsin-catabolized IGF1R. In lanes 13–17, the cells were treated as described for lanes 7–12, except that following trypsinization, cells were transferred to low-adherence plates, forcing cells to adapt to a state of anchorage-independence. The results confirmed the activity of cpd_W against the endogenous IRES in genetically-unmodified tumor cells. Similar results were obtained with IRES inhibitor lead cpd_P ( 11 ).
    Micrococcal Nuclease Treated Rabbit Reticulocyte Lysate, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eiken Chemical loopamp dna amplification kit
    Examination of stool samples weekly obtained from mice infected with 200 S. mansoni cercariae. (A) By using the <t>Loopamp</t> <t>DNA</t> amplification kit . (B) By using the SmMIT-LAMP developed in this study. Figure shows the results obtained in feces samples weekly obtained from week 0 p.i. to week 8 p.i. from an infected mouse randomly selected. Identical results were obtained in all infected mice. Lanes M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm, S. mansoni DNA, as positive control (1 ng); lanes 0–8, weeks 0, 1, 2, 3, 4, 5, 6, 7 and 8 p.i., respectively; lanes N; DNA mix from pooled DNA samples obtained from feces from non-infected mice, as negative control.
    Loopamp Dna Amplification Kit, supplied by Eiken Chemical, used in various techniques. Bioz Stars score: 94/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies brilliant ii sybr green qrt pcr master mix kit
    NCL does not significantly affect release of DENV particles. (A) Western blot of HEK293 cells infected with DENV (MOI, 3), followed by treatment with AS1411 or the NC. Cell supernatants were collected at time zero and at 24-h intervals until 96 h postinfection. Virus was purified through a sucrose cushion, and samples were analyzed by SDS-PAGE, followed by Western blotting of DENV C and DENV E proteins. The data are representative of three independent experiments. (B) <t>qRT-PCR</t> of viral RNA extracted from cell supernatants of DENV-infected HEK293 cells (as described for panel A) and no-virus control (NVC). Samples were analyzed using primers to DENV and normalized to norovirus G2 RNA added to the sample prior to extraction. The data are representative of two independent experiments performed in triplicate. (C) Western blot of HEK293 cells treated with siRNA to NCL or the NC, followed by infection with DENV (MOI, 3). Cell supernatants were collected at 72 h postinfection. Virus was purified through a sucrose cushion, and samples were analyzed by SDS-PAGE, followed by Western blotting of DENV C and DENV E proteins. (D) qRT-PCR of viral RNA extracted from cell supernatants of DENV-infected HEK293 cells after treatment with siRNA for NCL or negative-control siRNA. Samples were analyzed using primers to DENV and normalized to norovirus G2 RNA added to the sample prior to extraction. The data are representative of two independent experiments performed in triplicate. The error bars indicate SD.
    Brilliant Ii Sybr Green Qrt Pcr Master Mix Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 95/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Setting up the xCELLigence RTCA program schedule–Steps and Sweeps. An example of a Schedule page is shown for HEK293 cells. Step 1 (A) is preprogrammed and should not be changed. Use Add a Step and/or Add a Substep (A) to insert additional experimental steps with different sweeps and intervals in the schedule (B).

    Journal: Bio-protocol

    Article Title: Using xCELLigence RTCA Instrument to Measure Cell Adhesion

    doi: 10.21769/BioProtoc.2646

    Figure Lengend Snippet: Setting up the xCELLigence RTCA program schedule–Steps and Sweeps. An example of a Schedule page is shown for HEK293 cells. Step 1 (A) is preprogrammed and should not be changed. Use Add a Step and/or Add a Substep (A) to insert additional experimental steps with different sweeps and intervals in the schedule (B).

    Article Snippet: An example set up for HEK293 cells: Step 1 (background measurement); 1 sweep (total time 00:00:06).

    Techniques: Polyacrylamide Gel Electrophoresis

    Setting up the xCELLigence RTCA program–experimental details. An example of the Exp Notes (A) and the Layout page (B) for an experiment performed with HEK293 cells is shown. Four technical replicates are included for each condition. Details of the selected well in blue can be seen at the top of the page.

    Journal: Bio-protocol

    Article Title: Using xCELLigence RTCA Instrument to Measure Cell Adhesion

    doi: 10.21769/BioProtoc.2646

    Figure Lengend Snippet: Setting up the xCELLigence RTCA program–experimental details. An example of the Exp Notes (A) and the Layout page (B) for an experiment performed with HEK293 cells is shown. Four technical replicates are included for each condition. Details of the selected well in blue can be seen at the top of the page.

    Article Snippet: An example set up for HEK293 cells: Step 1 (background measurement); 1 sweep (total time 00:00:06).

    Techniques: Polyacrylamide Gel Electrophoresis

    Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed PCR. A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid DNA content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.

    Journal: Nature genetics

    Article Title: Haplotype-resolved whole genome sequencing by contiguity preserving transposition and combinatorial indexing

    doi: 10.1038/ng.3119

    Figure Lengend Snippet: Overview of the CPT-Seq workflow. There are three key steps: (I) indexed transposition, (II) pooling, diluting and compartmentalization, and (III) indexed PCR. A set of 96 different indexed transposome complexes are used to set up 96 independent transposition reactions to create separate genomic virtual partitions (step I). Transposition reactions are pooled together, diluted to sub-haploid DNA content, and split to 96 compartments (step II). Upon removal of the transposase with SDS, compartment-specific libraries are generated using indexed PCR (step III). All samples are pooled together after PCR, and prepared for sequencing.

    Article Snippet: 96 transposition reactions were set-up on ice in a low DNA-binding PCR plate (BioRad, Cat. No. HSS-9601).

    Techniques: Cycling Probe Technology, Polymerase Chain Reaction, Generated, Sequencing

    Tn5 transposase maintains contiguity of target DNA post-transposition.(a) PAGE-analysis of transposase contiguity: Tn5 transposome was used to target a ~1kb PCR amplicon. Transposed DNA was either treated with SDS to remove the transposase enzyme (lane 1), or as a control without SDS treatment (lane 2). Lane 3 is the input DNA and lane 4 is a 100bp reference ladder. As shown here, Tn5 transposase enzyme stays bound to its substrate DNA post-transposition and the protein-DNA complex only dissociates after addition of the protein denaturing agent, i.e., SDS. (b) Single molecule imaging of Tn5transposed DNA: HMW DNA (see Online methods ) labeled with YOYO-1 fluorescent dye was subjected to Tn5 transposition. SDS samples were treated with a final 0.05% SDS concentration and incubated at 55°C for 15 min.

    Journal: Nature genetics

    Article Title: Haplotype-resolved whole genome sequencing by contiguity preserving transposition and combinatorial indexing

    doi: 10.1038/ng.3119

    Figure Lengend Snippet: Tn5 transposase maintains contiguity of target DNA post-transposition.(a) PAGE-analysis of transposase contiguity: Tn5 transposome was used to target a ~1kb PCR amplicon. Transposed DNA was either treated with SDS to remove the transposase enzyme (lane 1), or as a control without SDS treatment (lane 2). Lane 3 is the input DNA and lane 4 is a 100bp reference ladder. As shown here, Tn5 transposase enzyme stays bound to its substrate DNA post-transposition and the protein-DNA complex only dissociates after addition of the protein denaturing agent, i.e., SDS. (b) Single molecule imaging of Tn5transposed DNA: HMW DNA (see Online methods ) labeled with YOYO-1 fluorescent dye was subjected to Tn5 transposition. SDS samples were treated with a final 0.05% SDS concentration and incubated at 55°C for 15 min.

    Article Snippet: 96 transposition reactions were set-up on ice in a low DNA-binding PCR plate (BioRad, Cat. No. HSS-9601).

    Techniques: Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Imaging, Labeling, Concentration Assay, Incubation

    ) or CVB3-0 (0) or cell culture medium (control). CVB3-binding isotype was detected by ELISA using peroxidase activity of bound goat anti-mouse IgG1 or bound goat anti-mouse IgG2a and quantitated by absorbance at 405 nm.

    Journal: Journal of Virology

    Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

    doi:

    Figure Lengend Snippet: ) or CVB3-0 (0) or cell culture medium (control). CVB3-binding isotype was detected by ELISA using peroxidase activity of bound goat anti-mouse IgG1 or bound goat anti-mouse IgG2a and quantitated by absorbance at 405 nm.

    Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

    Techniques: Cell Culture, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Characterization of mIL-4-expressing CVB3 strains in cell culture. (A) Single-step growth curves of the viruses on HeLa cells. (B) Concentration of mIL-4 in lysates as measured by ELISA. HeLa cells were inoculated with the parental virus CVB3/0 or the chimeric strains CVB3/0-mIL4-47 and CVB3-PL2-mIL4-46. Inoculated cell cultures were harvested by freeze-thaw lysis at the stated times. Error bars indicate standard error of the mean.

    Journal: Journal of Virology

    Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

    doi:

    Figure Lengend Snippet: Characterization of mIL-4-expressing CVB3 strains in cell culture. (A) Single-step growth curves of the viruses on HeLa cells. (B) Concentration of mIL-4 in lysates as measured by ELISA. HeLa cells were inoculated with the parental virus CVB3/0 or the chimeric strains CVB3/0-mIL4-47 and CVB3-PL2-mIL4-46. Inoculated cell cultures were harvested by freeze-thaw lysis at the stated times. Error bars indicate standard error of the mean.

    Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

    Techniques: Expressing, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lysis

    Western blot analysis of viral proteins in inoculated HeLa cells. Proteins were harvested from HeLa cells inoculated with the parental CVB3/0 or either of the chimeric strains by lysing the cell monolayers in Laemmli loading buffer. Lysates were prepared at the times shown (in hours p.i.) and electrophoresed in SDS–14% PAGE followed by electroblotting. Capsid protein 1D was detected with a polyclonal horse anti-CVB3 neutralizing antibody followed by peroxidase-conjugated rabbit anti-horse IgG. C, purified CVB3/0; TC, uninfected HeLa cell lysate. 1D, location of the detected capsid protein at 34 kDa is shown. Lane M, size markers (in kilodaltons).

    Journal: Journal of Virology

    Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

    doi:

    Figure Lengend Snippet: Western blot analysis of viral proteins in inoculated HeLa cells. Proteins were harvested from HeLa cells inoculated with the parental CVB3/0 or either of the chimeric strains by lysing the cell monolayers in Laemmli loading buffer. Lysates were prepared at the times shown (in hours p.i.) and electrophoresed in SDS–14% PAGE followed by electroblotting. Capsid protein 1D was detected with a polyclonal horse anti-CVB3 neutralizing antibody followed by peroxidase-conjugated rabbit anti-horse IgG. C, purified CVB3/0; TC, uninfected HeLa cell lysate. 1D, location of the detected capsid protein at 34 kDa is shown. Lane M, size markers (in kilodaltons).

    Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

    Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Purification

    RT-PCR analysis of mIL-4 insert stability following passage of chimeric viruses in HeLa cells. Amplimers were obtained and analyzed on 1.5% agarose gels following RT-PCR of viral RNA isolated from sequentially passaged CVB3-PL2-mIL4/46 (A and C) and CVB3/0-mIL4/47 (B and D). ELISA detection of mIL-4 protein is also shown for comparison (C and D). Viruses were passaged 10 times in HeLa cells as described in the text. Amplification of the region containing the insert in the viral RNA populations was accomplished with the primers ID9 and ID10 (A and B); the intact mIL-4 insert was detected as an 836-bp (or 830-bp) product and as a 344-bp product when the insert was deleted. Specific detection of the mIL-4-containing insert was accomplished using primers KNIL4S and KNIL4AS (C and D); the presence intact insert was detected as a 414-bp product, whereas deletion of the product resulted in no amplimer produced. mIL-4 ELISA (C and D), concentration of mIL-4 protein as detected by ELISA for each pass (5 × 10 5 HeLa cells infected at an MOI of ≥10 with designated viral passage stock, harvested at 6 h p.i.). Specific activities of mIL-4 ranged between 0.13 and 0.61 U/pg. Lanes P2 to P10, results from passages 2 to 10, respectively; lane M, 100-bp ladder (Life Technologies); lane PC, positive control amplification using pCVB3-PL2-mIL4/46 DNA; lane −, negative control (no DNA added) amplification; lane 0, PCR of CVB3/0 virus stock. Gel was stained with Cyber Green (FMC, Philadelphia, Pa.); images were captured using a Nucleo Vision gel documentation system (Nucleo Tech Corp.).

    Journal: Journal of Virology

    Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

    doi:

    Figure Lengend Snippet: RT-PCR analysis of mIL-4 insert stability following passage of chimeric viruses in HeLa cells. Amplimers were obtained and analyzed on 1.5% agarose gels following RT-PCR of viral RNA isolated from sequentially passaged CVB3-PL2-mIL4/46 (A and C) and CVB3/0-mIL4/47 (B and D). ELISA detection of mIL-4 protein is also shown for comparison (C and D). Viruses were passaged 10 times in HeLa cells as described in the text. Amplification of the region containing the insert in the viral RNA populations was accomplished with the primers ID9 and ID10 (A and B); the intact mIL-4 insert was detected as an 836-bp (or 830-bp) product and as a 344-bp product when the insert was deleted. Specific detection of the mIL-4-containing insert was accomplished using primers KNIL4S and KNIL4AS (C and D); the presence intact insert was detected as a 414-bp product, whereas deletion of the product resulted in no amplimer produced. mIL-4 ELISA (C and D), concentration of mIL-4 protein as detected by ELISA for each pass (5 × 10 5 HeLa cells infected at an MOI of ≥10 with designated viral passage stock, harvested at 6 h p.i.). Specific activities of mIL-4 ranged between 0.13 and 0.61 U/pg. Lanes P2 to P10, results from passages 2 to 10, respectively; lane M, 100-bp ladder (Life Technologies); lane PC, positive control amplification using pCVB3-PL2-mIL4/46 DNA; lane −, negative control (no DNA added) amplification; lane 0, PCR of CVB3/0 virus stock. Gel was stained with Cyber Green (FMC, Philadelphia, Pa.); images were captured using a Nucleo Vision gel documentation system (Nucleo Tech Corp.).

    Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay, Amplification, Produced, Concentration Assay, Infection, Positive Control, Negative Control, Polymerase Chain Reaction, Staining

    Detection of CVB3-associated mIL-4 insert at 14 days p.i. in mice. Amplimers were obtained and analyzed on 1.5% agarose gels following RT-PCR of viral RNA in total tissue RNA isolated from hearts (A), pancreas (B), and liver (C) of mice inoculated 14 days previously with 5 × 10 5 TCID 50 of CVB3-PL2-mIL4/46. cDNA was made from RNA of tissues from five individual mice. Amplification of the region containing the insert in the viral RNA populations was accomplished using the CVB3-based primers ID9 and ID10. The intact mIL-4 insert was detected as an 830-bp product and as a 344-bp product when the insert was deleted. Lanes 1 to 5, PCR from cDNA of individual mouse tissues; lane 46, PCR from CVB3-PL2-mIL4/46 virus stock; lane 0, PCR from CVB3/0 virus stock; lane M, 100-bp ladder (Life Technologies). Gel was stained with Cyber Green (FMC) and images were captured using a Nucleo Vision gel documentation system (Nucleo Tech Corp.).

    Journal: Journal of Virology

    Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

    doi:

    Figure Lengend Snippet: Detection of CVB3-associated mIL-4 insert at 14 days p.i. in mice. Amplimers were obtained and analyzed on 1.5% agarose gels following RT-PCR of viral RNA in total tissue RNA isolated from hearts (A), pancreas (B), and liver (C) of mice inoculated 14 days previously with 5 × 10 5 TCID 50 of CVB3-PL2-mIL4/46. cDNA was made from RNA of tissues from five individual mice. Amplification of the region containing the insert in the viral RNA populations was accomplished using the CVB3-based primers ID9 and ID10. The intact mIL-4 insert was detected as an 830-bp product and as a 344-bp product when the insert was deleted. Lanes 1 to 5, PCR from cDNA of individual mouse tissues; lane 46, PCR from CVB3-PL2-mIL4/46 virus stock; lane 0, PCR from CVB3/0 virus stock; lane M, 100-bp ladder (Life Technologies). Gel was stained with Cyber Green (FMC) and images were captured using a Nucleo Vision gel documentation system (Nucleo Tech Corp.).

    Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

    Techniques: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Polymerase Chain Reaction, Staining

    The expression of RAD51 allele from various pYNRAD51 plasmids. ( A ) RT–PCR analysis of total RNA extracts from LSY678(Int) strain harboring various pYNRAD51 mutant plasmids. A 1.2 kb RAD51 product was amplified using primers of Rad51F and Rad51B and electrophoresed through 1% agarose gel. ( B ) Western blot analysis of whole-cell extracts from LSY402(Δrad51) and LSY678(Int) containing the various pYNRAD51 expression plasmids. Protein extracts from LSY402(Δrad51) or LSY678(Int), alone or containing the different pYNRAD51 expression plasmids, were evaluated by blotting with rabbit α Rad51 antibody and visualized by an anti-rabbit antibody conjugated with HRP in an enhanced chemiluminescence ECL™ system. Protein extracts from LSY402(Δrad51) with empty vector (pYN132) were used as a negative control. An internal control was set up by utilizing a goat β-actin antibody and visualized by anti-goat IgG-HRP antibody in the same ECL™ system.

    Journal: Nucleic Acids Research

    Article Title: Genetic re-engineering of Saccharomyces cerevisiae RAD51 leads to a significant increase in the frequency of gene repair in vivo

    doi: 10.1093/nar/gkh506

    Figure Lengend Snippet: The expression of RAD51 allele from various pYNRAD51 plasmids. ( A ) RT–PCR analysis of total RNA extracts from LSY678(Int) strain harboring various pYNRAD51 mutant plasmids. A 1.2 kb RAD51 product was amplified using primers of Rad51F and Rad51B and electrophoresed through 1% agarose gel. ( B ) Western blot analysis of whole-cell extracts from LSY402(Δrad51) and LSY678(Int) containing the various pYNRAD51 expression plasmids. Protein extracts from LSY402(Δrad51) or LSY678(Int), alone or containing the different pYNRAD51 expression plasmids, were evaluated by blotting with rabbit α Rad51 antibody and visualized by an anti-rabbit antibody conjugated with HRP in an enhanced chemiluminescence ECL™ system. Protein extracts from LSY402(Δrad51) with empty vector (pYN132) were used as a negative control. An internal control was set up by utilizing a goat β-actin antibody and visualized by anti-goat IgG-HRP antibody in the same ECL™ system.

    Article Snippet: An internal control was set up by utilizing a goat β-actin antibody (Santa Cruz Biotech Inc., Santa Cruz, CA) in a 1:500 dilution and visualized by anti-goat IgG-HRP (Sigma, St Louis, MO) in a dilution of 1:500 in the same ECL™ developing system.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Amplification, Agarose Gel Electrophoresis, Western Blot, Plasmid Preparation, Negative Control

    Mass spectrometry indicates that human CM lactoferrin binds DC-SIGN. (A) 4–12% SDS PAGE gel loaded (from left to right) with a 250kD protein marker, agarose beads, DC-SIGN-Fc, the supernatant from the first wash, supernatant from the second wash, and the DC-SIGN-Fc coated agarose beads loaded with the DC-SIGN binding component from CM. Band #2, 3 and 4 potentially contain the DC-SIGN binding component of CM. Ion trap mass spectrometry of in gel digests identified human lactoferrin fragments (highly abundant in band #3) and immunoglobulins in all three bands and intelectin-1 in band #4 (with trace amounts in the other bands). (B) CM representing a high DC-SIGN binder, an intermediate DC-SIGN binder and a low/no DC-SIGN binder were coated on an ELISA plate and were tested for DC-SIGN and lactoferrin binding. The first graph (left) confirms the DC-SIGN binding status while the second graph (right) shows the binding capacity of polyclonal anti-lactoferrin, which is high for CM from a DC-SIGN high binder, intermediate for an intermediate DC-SIGN binder and not present in CM from a low/no DC-SIGN binder.

    Journal: PLoS ONE

    Article Title: Colorectal Mucus Binds DC-SIGN and Inhibits HIV-1 Trans-Infection of CD4+ T-Lymphocytes

    doi: 10.1371/journal.pone.0122020

    Figure Lengend Snippet: Mass spectrometry indicates that human CM lactoferrin binds DC-SIGN. (A) 4–12% SDS PAGE gel loaded (from left to right) with a 250kD protein marker, agarose beads, DC-SIGN-Fc, the supernatant from the first wash, supernatant from the second wash, and the DC-SIGN-Fc coated agarose beads loaded with the DC-SIGN binding component from CM. Band #2, 3 and 4 potentially contain the DC-SIGN binding component of CM. Ion trap mass spectrometry of in gel digests identified human lactoferrin fragments (highly abundant in band #3) and immunoglobulins in all three bands and intelectin-1 in band #4 (with trace amounts in the other bands). (B) CM representing a high DC-SIGN binder, an intermediate DC-SIGN binder and a low/no DC-SIGN binder were coated on an ELISA plate and were tested for DC-SIGN and lactoferrin binding. The first graph (left) confirms the DC-SIGN binding status while the second graph (right) shows the binding capacity of polyclonal anti-lactoferrin, which is high for CM from a DC-SIGN high binder, intermediate for an intermediate DC-SIGN binder and not present in CM from a low/no DC-SIGN binder.

    Article Snippet: The same set up was used for detecting lactoferrin and intelectin-1 in CM, only instead of DC-SIGN-Fc, 333ng/ml polyclonal anti-lactoferrin (ab15811, Abcam) and 333ng/ml anti-intelectin-1 (ab118232, Abcam) was used.

    Techniques: Mass Spectrometry, SDS Page, Marker, Binding Assay, Enzyme-linked Immunosorbent Assay

    IRES-mediated translation and IRES inhibition in a cell-free system and in cells. (A) Diagrammatic comparison of general protein synthesis to IRES-mediated translation. General protein synthesis is mediated by cap-dependent ribosomal scanning from the 5′-end of the mRNA and may be modulated by mTOR inhibitors. Internal ribosome entry sites (IRESs) allow the 40S ribosome to engage the mRNA at a position much closer (in many cases immediately adjacent to) the AUG initiation codon. IRES-mediated translation is independently regulated and serves as a fail-safe mechanism ensuring the synthesis of proteins most critical for cell survival. (B) Structure of IRES inhibitor lead compound W (cpd_W): Ethyl 2-{[2-(1,3-benzoxazol-2-ylthio)butanoyl]amino}-4-methyl-1,3-thiazole-5-carboxylate, MW 405. (C) In vitro translation assays: Rabbit reticulocyte lysate was programmed with a bicistronic reporter RNA in which translation of the second cistron (firefly luciferase coding sequence) is mediated by the IGF1R IRES, while translation of the first cistron ( Renilla luciferase coding sequence) is mediated by ribosomal scanning. IRES inhibitor cpd_W (or vehicle control) was included in the reaction in increasing concentrations as indicated. The result is indicative of selective inhibition of IRES-mediated translation. A structural analog of cpd_W (W-7) in which a single atom has been modified (converting the benzoxazole to a benzimidazole) was completely inactive in this assay, indicative of the chemical specificity of IRES inhibition. Cycloheximide (5 µg/ml, chx) and puromycin (250 µg/ml, puro) were included as reference standards for non-specific translational inhibition (far right). (D) IRES inhibitor cpd_W completely blocked de novo synthesis of IGF1R in breast tumor cells under adverse conditions (serum-deprivation, loss of adhesion) relevant to the microenvironment of the tumor. T47D breast tumor cells were seeded in 6-well plates and allowed 48 h to recover and resume proliferation, then incubated in the presence of IRES inhibitor cpd_W (10 µg/ml) or vehicle control (0.1% DMSO) as indicated. The cells were simultaneously subjected to acute serum deprivation (0.5% fetal calf serum, no added insulin) to increase dependence on IRES-mediated translation. After 24 h, the cells were harvested and whole cell lysates prepared, equivalent aliquots separated by SDS-PAGE and immunoblotted for IGF1R-β and α-tubulin. In lanes 7–12, the cells were trypsinized and seeded into 6-well plates and immediately incubated in the presence of IRES-inhibitor cpd_W or vehicle control as indicated. Robust regeneration of trypsin-catabolized IGF1R was observed within 24 h in vehicle-treated cells, however, this was completely blocked in the presence of cpd_W (10 µg/ml as shown; IC 50 , 2 µg/ml). The asterisk (*) marks the position of trypsin-catabolized IGF1R. In lanes 13–17, the cells were treated as described for lanes 7–12, except that following trypsinization, cells were transferred to low-adherence plates, forcing cells to adapt to a state of anchorage-independence. The results confirmed the activity of cpd_W against the endogenous IRES in genetically-unmodified tumor cells. Similar results were obtained with IRES inhibitor lead cpd_P ( 11 ).

    Journal: Oncology Reports

    Article Title: Translational control of the undifferentiated phenotype in ER-positive breast tumor cells: Cytoplasmic localization of ERα and impact of IRES inhibition

    doi: 10.3892/or.2018.6332

    Figure Lengend Snippet: IRES-mediated translation and IRES inhibition in a cell-free system and in cells. (A) Diagrammatic comparison of general protein synthesis to IRES-mediated translation. General protein synthesis is mediated by cap-dependent ribosomal scanning from the 5′-end of the mRNA and may be modulated by mTOR inhibitors. Internal ribosome entry sites (IRESs) allow the 40S ribosome to engage the mRNA at a position much closer (in many cases immediately adjacent to) the AUG initiation codon. IRES-mediated translation is independently regulated and serves as a fail-safe mechanism ensuring the synthesis of proteins most critical for cell survival. (B) Structure of IRES inhibitor lead compound W (cpd_W): Ethyl 2-{[2-(1,3-benzoxazol-2-ylthio)butanoyl]amino}-4-methyl-1,3-thiazole-5-carboxylate, MW 405. (C) In vitro translation assays: Rabbit reticulocyte lysate was programmed with a bicistronic reporter RNA in which translation of the second cistron (firefly luciferase coding sequence) is mediated by the IGF1R IRES, while translation of the first cistron ( Renilla luciferase coding sequence) is mediated by ribosomal scanning. IRES inhibitor cpd_W (or vehicle control) was included in the reaction in increasing concentrations as indicated. The result is indicative of selective inhibition of IRES-mediated translation. A structural analog of cpd_W (W-7) in which a single atom has been modified (converting the benzoxazole to a benzimidazole) was completely inactive in this assay, indicative of the chemical specificity of IRES inhibition. Cycloheximide (5 µg/ml, chx) and puromycin (250 µg/ml, puro) were included as reference standards for non-specific translational inhibition (far right). (D) IRES inhibitor cpd_W completely blocked de novo synthesis of IGF1R in breast tumor cells under adverse conditions (serum-deprivation, loss of adhesion) relevant to the microenvironment of the tumor. T47D breast tumor cells were seeded in 6-well plates and allowed 48 h to recover and resume proliferation, then incubated in the presence of IRES inhibitor cpd_W (10 µg/ml) or vehicle control (0.1% DMSO) as indicated. The cells were simultaneously subjected to acute serum deprivation (0.5% fetal calf serum, no added insulin) to increase dependence on IRES-mediated translation. After 24 h, the cells were harvested and whole cell lysates prepared, equivalent aliquots separated by SDS-PAGE and immunoblotted for IGF1R-β and α-tubulin. In lanes 7–12, the cells were trypsinized and seeded into 6-well plates and immediately incubated in the presence of IRES-inhibitor cpd_W or vehicle control as indicated. Robust regeneration of trypsin-catabolized IGF1R was observed within 24 h in vehicle-treated cells, however, this was completely blocked in the presence of cpd_W (10 µg/ml as shown; IC 50 , 2 µg/ml). The asterisk (*) marks the position of trypsin-catabolized IGF1R. In lanes 13–17, the cells were treated as described for lanes 7–12, except that following trypsinization, cells were transferred to low-adherence plates, forcing cells to adapt to a state of anchorage-independence. The results confirmed the activity of cpd_W against the endogenous IRES in genetically-unmodified tumor cells. Similar results were obtained with IRES inhibitor lead cpd_P ( 11 ).

    Article Snippet: In vitro translation Standard in vitro translation reactions were set up using micrococcal nuclease treated rabbit reticulocyte lysate (Promega) at a final concentration of 50% (vol/vol).

    Techniques: Inhibition, In Vitro, Luciferase, Sequencing, Modification, Incubation, SDS Page, Activity Assay

    Examination of stool samples weekly obtained from mice infected with 200 S. mansoni cercariae. (A) By using the Loopamp DNA amplification kit . (B) By using the SmMIT-LAMP developed in this study. Figure shows the results obtained in feces samples weekly obtained from week 0 p.i. to week 8 p.i. from an infected mouse randomly selected. Identical results were obtained in all infected mice. Lanes M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm, S. mansoni DNA, as positive control (1 ng); lanes 0–8, weeks 0, 1, 2, 3, 4, 5, 6, 7 and 8 p.i., respectively; lanes N; DNA mix from pooled DNA samples obtained from feces from non-infected mice, as negative control.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

    doi: 10.1371/journal.pntd.0003126

    Figure Lengend Snippet: Examination of stool samples weekly obtained from mice infected with 200 S. mansoni cercariae. (A) By using the Loopamp DNA amplification kit . (B) By using the SmMIT-LAMP developed in this study. Figure shows the results obtained in feces samples weekly obtained from week 0 p.i. to week 8 p.i. from an infected mouse randomly selected. Identical results were obtained in all infected mice. Lanes M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm, S. mansoni DNA, as positive control (1 ng); lanes 0–8, weeks 0, 1, 2, 3, 4, 5, 6, 7 and 8 p.i., respectively; lanes N; DNA mix from pooled DNA samples obtained from feces from non-infected mice, as negative control.

    Article Snippet: Setting up LAMP assay The optimal incubation temperature for LAMP assay using the Loopamp DNA amplification Kit tested with the S. mansoni primer set was established in a conventional heating block using a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5 min to inactivate the enzyme.

    Techniques: Mouse Assay, Infection, Amplification, Molecular Weight, Marker, Positive Control, Negative Control

    LAMP detection of S. mansoni genomic DNA samples using SmMIT-LAMP or the Loopamp DNA amplification kit at 63°C for 1 h. (A) The turbidity of the reaction mixture was inspected by the naked eye. (B) The LAMP amplification results were also visually detected by adding the fluorescent dye SYBR Green I to the reaction tubes. A successful LAMP reaction would turn to green; otherwise, it would remain orange (C) LAMP products were also monitored using 2% agarose gel electrophoresis stained with ethidium bromide. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm: S. mansoni DNA (1 ng); lane N, negative control (no DNA template).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

    doi: 10.1371/journal.pntd.0003126

    Figure Lengend Snippet: LAMP detection of S. mansoni genomic DNA samples using SmMIT-LAMP or the Loopamp DNA amplification kit at 63°C for 1 h. (A) The turbidity of the reaction mixture was inspected by the naked eye. (B) The LAMP amplification results were also visually detected by adding the fluorescent dye SYBR Green I to the reaction tubes. A successful LAMP reaction would turn to green; otherwise, it would remain orange (C) LAMP products were also monitored using 2% agarose gel electrophoresis stained with ethidium bromide. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm: S. mansoni DNA (1 ng); lane N, negative control (no DNA template).

    Article Snippet: Setting up LAMP assay The optimal incubation temperature for LAMP assay using the Loopamp DNA amplification Kit tested with the S. mansoni primer set was established in a conventional heating block using a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5 min to inactivate the enzyme.

    Techniques: Amplification, SYBR Green Assay, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker, Negative Control

    Specificity and sensitivity assessment of the LAMP assay for S. mansoni . (A) Specificity assessment performed with SmMIT-LAMP is shown. Identical results were obtained using Loopamp DNA amplification Kit . A ladder of multiple bands of different sizes could be only observed in S. mansoni DNA sample. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm, Sh, Sj, Si, Fh, Dd, Hd, Cd, Ll, Bp, As, Ts, Tt, Eg, Cp, Gd, Eh, S. mansoni , S. haematobium , S. japonicum , S. intercalatum , Dicrocoelium dendriticum , Hymenolepis diminuta , Calicophoron daubneyi , Loa loa , Brugia pahangi , Anisakis simplex , Trichinella spiralis , Taenia taeniformis , Echinococcus granulosus , Cryptosporidium parvum , Giardia intestinalis and Entamoeba histolytica DNA samples (1 ng/each), respectively; lane N, negative control (no DNA template). (B) Sensitivity assessment performed with the Loopamp DNA amplification kit at 63°C for 1 h using serial dilutions of S. mansoni genomic DNA by the addition of SYBR Green I (up) or by visualization on agarose gel (down). (C) Sensitivity assessment performed with SmMIT-LAMP at 63°C for 1 h using serial dilutions of S. mansoni genomic DNA by the addition of SYBR Green I (up) or by visualization on agarose gel (down). Lane M: 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm: genomic DNA from S. mansoni (1 ng); lanes 10 −1 –10 −9 : 10-fold serially dilutions; lanes N: negative controls (no DNA template).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

    doi: 10.1371/journal.pntd.0003126

    Figure Lengend Snippet: Specificity and sensitivity assessment of the LAMP assay for S. mansoni . (A) Specificity assessment performed with SmMIT-LAMP is shown. Identical results were obtained using Loopamp DNA amplification Kit . A ladder of multiple bands of different sizes could be only observed in S. mansoni DNA sample. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm, Sh, Sj, Si, Fh, Dd, Hd, Cd, Ll, Bp, As, Ts, Tt, Eg, Cp, Gd, Eh, S. mansoni , S. haematobium , S. japonicum , S. intercalatum , Dicrocoelium dendriticum , Hymenolepis diminuta , Calicophoron daubneyi , Loa loa , Brugia pahangi , Anisakis simplex , Trichinella spiralis , Taenia taeniformis , Echinococcus granulosus , Cryptosporidium parvum , Giardia intestinalis and Entamoeba histolytica DNA samples (1 ng/each), respectively; lane N, negative control (no DNA template). (B) Sensitivity assessment performed with the Loopamp DNA amplification kit at 63°C for 1 h using serial dilutions of S. mansoni genomic DNA by the addition of SYBR Green I (up) or by visualization on agarose gel (down). (C) Sensitivity assessment performed with SmMIT-LAMP at 63°C for 1 h using serial dilutions of S. mansoni genomic DNA by the addition of SYBR Green I (up) or by visualization on agarose gel (down). Lane M: 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm: genomic DNA from S. mansoni (1 ng); lanes 10 −1 –10 −9 : 10-fold serially dilutions; lanes N: negative controls (no DNA template).

    Article Snippet: Setting up LAMP assay The optimal incubation temperature for LAMP assay using the Loopamp DNA amplification Kit tested with the S. mansoni primer set was established in a conventional heating block using a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5 min to inactivate the enzyme.

    Techniques: Lamp Assay, Amplification, Molecular Weight, Marker, Negative Control, SYBR Green Assay, Agarose Gel Electrophoresis

    NCL does not significantly affect release of DENV particles. (A) Western blot of HEK293 cells infected with DENV (MOI, 3), followed by treatment with AS1411 or the NC. Cell supernatants were collected at time zero and at 24-h intervals until 96 h postinfection. Virus was purified through a sucrose cushion, and samples were analyzed by SDS-PAGE, followed by Western blotting of DENV C and DENV E proteins. The data are representative of three independent experiments. (B) qRT-PCR of viral RNA extracted from cell supernatants of DENV-infected HEK293 cells (as described for panel A) and no-virus control (NVC). Samples were analyzed using primers to DENV and normalized to norovirus G2 RNA added to the sample prior to extraction. The data are representative of two independent experiments performed in triplicate. (C) Western blot of HEK293 cells treated with siRNA to NCL or the NC, followed by infection with DENV (MOI, 3). Cell supernatants were collected at 72 h postinfection. Virus was purified through a sucrose cushion, and samples were analyzed by SDS-PAGE, followed by Western blotting of DENV C and DENV E proteins. (D) qRT-PCR of viral RNA extracted from cell supernatants of DENV-infected HEK293 cells after treatment with siRNA for NCL or negative-control siRNA. Samples were analyzed using primers to DENV and normalized to norovirus G2 RNA added to the sample prior to extraction. The data are representative of two independent experiments performed in triplicate. The error bars indicate SD.

    Journal: Journal of Virology

    Article Title: Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

    doi: 10.1128/JVI.00704-13

    Figure Lengend Snippet: NCL does not significantly affect release of DENV particles. (A) Western blot of HEK293 cells infected with DENV (MOI, 3), followed by treatment with AS1411 or the NC. Cell supernatants were collected at time zero and at 24-h intervals until 96 h postinfection. Virus was purified through a sucrose cushion, and samples were analyzed by SDS-PAGE, followed by Western blotting of DENV C and DENV E proteins. The data are representative of three independent experiments. (B) qRT-PCR of viral RNA extracted from cell supernatants of DENV-infected HEK293 cells (as described for panel A) and no-virus control (NVC). Samples were analyzed using primers to DENV and normalized to norovirus G2 RNA added to the sample prior to extraction. The data are representative of two independent experiments performed in triplicate. (C) Western blot of HEK293 cells treated with siRNA to NCL or the NC, followed by infection with DENV (MOI, 3). Cell supernatants were collected at 72 h postinfection. Virus was purified through a sucrose cushion, and samples were analyzed by SDS-PAGE, followed by Western blotting of DENV C and DENV E proteins. (D) qRT-PCR of viral RNA extracted from cell supernatants of DENV-infected HEK293 cells after treatment with siRNA for NCL or negative-control siRNA. Samples were analyzed using primers to DENV and normalized to norovirus G2 RNA added to the sample prior to extraction. The data are representative of two independent experiments performed in triplicate. The error bars indicate SD.

    Article Snippet: Quantitative real-time PCRs (qRT-PCRs) were set up using the Brilliant II SYBR green QRT-PCR Master Mix Kit, 1-step (Agilent Technologies, Santa Clara, CA).

    Techniques: Western Blot, Infection, Purification, SDS Page, Quantitative RT-PCR, Negative Control

    NCL affects DENV capsid migration characteristics. HEK293 cells were infected with DENV (MOI, 3), followed by treatment with AS1411 or the negative control. Cell supernatants were collected at 72 h postinfection, and virus was purified through a sucrose cushion. (A) Sucrose-purified virus was examined by SDS-PAGE, followed by Western blotting for DENV C and E proteins. (B) The sucrose-purified samples were also incubated for 10 min at the indicated temperatures and run on a native AGE gel. Samples were analyzed by Western blotting for DENV C protein. (C) qRT-PCR of sucrose-purified virus after incubation at the indicated temperatures and NVC. Viral RNA was detected by qRT-PCR using primers to DENV and normalized to the 4°C input samples. The data are representative of two independent experiments performed in triplicate. The error bars indicate SD.

    Journal: Journal of Virology

    Article Title: Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

    doi: 10.1128/JVI.00704-13

    Figure Lengend Snippet: NCL affects DENV capsid migration characteristics. HEK293 cells were infected with DENV (MOI, 3), followed by treatment with AS1411 or the negative control. Cell supernatants were collected at 72 h postinfection, and virus was purified through a sucrose cushion. (A) Sucrose-purified virus was examined by SDS-PAGE, followed by Western blotting for DENV C and E proteins. (B) The sucrose-purified samples were also incubated for 10 min at the indicated temperatures and run on a native AGE gel. Samples were analyzed by Western blotting for DENV C protein. (C) qRT-PCR of sucrose-purified virus after incubation at the indicated temperatures and NVC. Viral RNA was detected by qRT-PCR using primers to DENV and normalized to the 4°C input samples. The data are representative of two independent experiments performed in triplicate. The error bars indicate SD.

    Article Snippet: Quantitative real-time PCRs (qRT-PCRs) were set up using the Brilliant II SYBR green QRT-PCR Master Mix Kit, 1-step (Agilent Technologies, Santa Clara, CA).

    Techniques: Migration, Infection, Negative Control, Purification, SDS Page, Western Blot, Incubation, Quantitative RT-PCR

    NCL interaction with DENV C is RNA independent. (A) Co-IP of HEK293 cells transfected with either GFP-DVC or GFP-CAT. Cell lysates were treated with RNase A or left untreated, and co-IP was performed using αGFP antibody as previously described. Western blots were stained using antibodies to NCL, PABP, or GFP. The Western blots are representative of three independent experiments. (B) RNA-IP of HEK293 cells transfected with GFP-NCL, GFP-PABP, or GFP-CAT expression vectors. Co-IP was performed using αGFP antibody, and RNA was extracted from the input or co-IP sample. Samples were analyzed by qRT-PCR using DENV forward and reverse primers. The data are representative of two independent experiments performed in triplicate. dRn, change in the normalized reporter signal.

    Journal: Journal of Virology

    Article Title: Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

    doi: 10.1128/JVI.00704-13

    Figure Lengend Snippet: NCL interaction with DENV C is RNA independent. (A) Co-IP of HEK293 cells transfected with either GFP-DVC or GFP-CAT. Cell lysates were treated with RNase A or left untreated, and co-IP was performed using αGFP antibody as previously described. Western blots were stained using antibodies to NCL, PABP, or GFP. The Western blots are representative of three independent experiments. (B) RNA-IP of HEK293 cells transfected with GFP-NCL, GFP-PABP, or GFP-CAT expression vectors. Co-IP was performed using αGFP antibody, and RNA was extracted from the input or co-IP sample. Samples were analyzed by qRT-PCR using DENV forward and reverse primers. The data are representative of two independent experiments performed in triplicate. dRn, change in the normalized reporter signal.

    Article Snippet: Quantitative real-time PCRs (qRT-PCRs) were set up using the Brilliant II SYBR green QRT-PCR Master Mix Kit, 1-step (Agilent Technologies, Santa Clara, CA).

    Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Staining, Expressing, Quantitative RT-PCR

    NCL does not significantly affect DENV RNA replication or protein translation. (A) qRT-PCR of viral RNA extracted from DENV-infected (MOI, 3) HEK293 cells after treatment with AS1411. Samples were analyzed using primers to DENV and normalized to GAPDH. (B) Western blot of cell lysates collected from DENV-infected cells, followed by treatment with AS1411 or the NC and collection at the indicated time points. The Western blots were stained with antibodies to the DENV C and DENV E proteins, as well as with actin. The Western blots are representative of three independent experiments. (C) qRT-PCR of viral RNA extracted from HEK293 cells treated with siRNA to NCL or nonspecific siRNA, followed by infection with DENV (MOI, 3) for 72 h. Samples were analyzed using primers to DENV and normalized to GAPDH. (D) Western blot of HEK293 cells treated with siRNA to NCL or the NC, followed by infection with DENV (MOI, 3). Samples were collected at 72 h postinfection and examined by Western blotting for NCL, DENV C, DENV E, and actin. The error bars indicate SD.

    Journal: Journal of Virology

    Article Title: Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

    doi: 10.1128/JVI.00704-13

    Figure Lengend Snippet: NCL does not significantly affect DENV RNA replication or protein translation. (A) qRT-PCR of viral RNA extracted from DENV-infected (MOI, 3) HEK293 cells after treatment with AS1411. Samples were analyzed using primers to DENV and normalized to GAPDH. (B) Western blot of cell lysates collected from DENV-infected cells, followed by treatment with AS1411 or the NC and collection at the indicated time points. The Western blots were stained with antibodies to the DENV C and DENV E proteins, as well as with actin. The Western blots are representative of three independent experiments. (C) qRT-PCR of viral RNA extracted from HEK293 cells treated with siRNA to NCL or nonspecific siRNA, followed by infection with DENV (MOI, 3) for 72 h. Samples were analyzed using primers to DENV and normalized to GAPDH. (D) Western blot of HEK293 cells treated with siRNA to NCL or the NC, followed by infection with DENV (MOI, 3). Samples were collected at 72 h postinfection and examined by Western blotting for NCL, DENV C, DENV E, and actin. The error bars indicate SD.

    Article Snippet: Quantitative real-time PCRs (qRT-PCRs) were set up using the Brilliant II SYBR green QRT-PCR Master Mix Kit, 1-step (Agilent Technologies, Santa Clara, CA).

    Techniques: Quantitative RT-PCR, Infection, Western Blot, Staining