pcdna dest40  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pcdna dest40
    Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. <t>pcDNA-DEST40</t> expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P
    Pcdna Dest40, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 33 article reviews
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    pcdna dest40 - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions"

    Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions

    Journal: Genomics, Proteomics & Bioinformatics

    doi: 10.1016/j.gpb.2018.12.010

    Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. pcDNA-DEST40 expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P
    Figure Legend Snippet: Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. pcDNA-DEST40 expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P

    Techniques Used: Expressing, Transfection, Immunoprecipitation, Plasmid Preparation, SDS Page, Infection, Staining, Real-time Polymerase Chain Reaction

    Identification of protein binders for BETi JQ1 by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated JQ1 (Bio-JQ1) relative to free biotin (Bio). 12,492 ORFs were ranked according to the ratio of Bio-JQ1/Bio (from the smallest to the largest). The red line shows the cut-off value. A known protein target of JQ1, BRD2, was ranked as a top hit (red dot). B. Hit validation of JQ1 protein targets identified from the PLATO-BC assay. pET-DEST42-EWSR1/EYA3/RBM14/BD1/BD2 was transformed into the E. coli DE3 strain. V5-His6-tagged EWSR1/EYA3/RBM14/BD1/BD2 proteins were purified and subjected to protein pull-down assays for Bio-JQ1 or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. C. Interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and subjected to protein pull-down assays for biotinylated, acetylated or non-acetylated H4 (Bio-H4ac or Bio-H4) peptide. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. D. Interaction of EWSR1 with H4ac in cells. pcDNA-DEST40-EWSR1 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-His6-tagged EWSR1 using an anti-V5 mouse antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-H4ac antibody. E. JQ1 interrupts the interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and incubated with JQ1 (at 1 μM or 10 μM) or DMSO, prior to the protein pull-down assays for Bio-H4ac or free biotin (Bio). Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody or HRP-conjugated streptavidin to detect EWSR1 or H4ac respectively. BETi, bromodomain and extraterminal domain inhibitor; BRD2, bromodomain containing 2; H4ac, acetylated histone H4; EWSR1, Ewing sarcoma breakpoint region 1; EYA3, EYA transcriptional coactivator and phosphatase 3; RBM14, RNA-binding protein 14; BD1, bromodomain 1; BD2, bromodomain 2.
    Figure Legend Snippet: Identification of protein binders for BETi JQ1 by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated JQ1 (Bio-JQ1) relative to free biotin (Bio). 12,492 ORFs were ranked according to the ratio of Bio-JQ1/Bio (from the smallest to the largest). The red line shows the cut-off value. A known protein target of JQ1, BRD2, was ranked as a top hit (red dot). B. Hit validation of JQ1 protein targets identified from the PLATO-BC assay. pET-DEST42-EWSR1/EYA3/RBM14/BD1/BD2 was transformed into the E. coli DE3 strain. V5-His6-tagged EWSR1/EYA3/RBM14/BD1/BD2 proteins were purified and subjected to protein pull-down assays for Bio-JQ1 or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. C. Interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and subjected to protein pull-down assays for biotinylated, acetylated or non-acetylated H4 (Bio-H4ac or Bio-H4) peptide. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. D. Interaction of EWSR1 with H4ac in cells. pcDNA-DEST40-EWSR1 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-His6-tagged EWSR1 using an anti-V5 mouse antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-H4ac antibody. E. JQ1 interrupts the interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and incubated with JQ1 (at 1 μM or 10 μM) or DMSO, prior to the protein pull-down assays for Bio-H4ac or free biotin (Bio). Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody or HRP-conjugated streptavidin to detect EWSR1 or H4ac respectively. BETi, bromodomain and extraterminal domain inhibitor; BRD2, bromodomain containing 2; H4ac, acetylated histone H4; EWSR1, Ewing sarcoma breakpoint region 1; EYA3, EYA transcriptional coactivator and phosphatase 3; RBM14, RNA-binding protein 14; BD1, bromodomain 1; BD2, bromodomain 2.

    Techniques Used: Positron Emission Tomography, Transformation Assay, Purification, SDS Page, In Vitro, Transfection, Immunoprecipitation, Incubation, RNA Binding Assay

    2) Product Images from "Use of a microscope stage-mounted Nickel-63 microirradiator for real-time observation of the DNA double-strand break response"

    Article Title: Use of a microscope stage-mounted Nickel-63 microirradiator for real-time observation of the DNA double-strand break response

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq409

    Calibration EYFP-53BP1 reporter system. ( A ) EYFP-53BP1 primary structures. EYFP coding sequence is joined to full-length 53BP1, with Tudor and BRCT domains indicated. EYFP1-53BP coding sequence was inserted into pcDNA-DEST40 (Invitrogen) for expression under control of the CMV promoter. ( B ) Dose response to calibrated doses of 137 Cs reference radiation. Cells were co-transfected with EYFP-53BP1 WT and H2B-diHcRed. Live-cell images were collected 30 min post-irradiation. ( C ) Quantification of dose response. Foci were scored using 8–24 individual nuclei at each dose point, and linear regression was performed to obtain the slope of the dose-response curve.
    Figure Legend Snippet: Calibration EYFP-53BP1 reporter system. ( A ) EYFP-53BP1 primary structures. EYFP coding sequence is joined to full-length 53BP1, with Tudor and BRCT domains indicated. EYFP1-53BP coding sequence was inserted into pcDNA-DEST40 (Invitrogen) for expression under control of the CMV promoter. ( B ) Dose response to calibrated doses of 137 Cs reference radiation. Cells were co-transfected with EYFP-53BP1 WT and H2B-diHcRed. Live-cell images were collected 30 min post-irradiation. ( C ) Quantification of dose response. Foci were scored using 8–24 individual nuclei at each dose point, and linear regression was performed to obtain the slope of the dose-response curve.

    Techniques Used: Sequencing, Expressing, Transfection, Irradiation

    3) Product Images from "R-Spondin Potentiates Wnt/?-Catenin Signaling through Orphan Receptors LGR4 and LGR5"

    Article Title: R-Spondin Potentiates Wnt/?-Catenin Signaling through Orphan Receptors LGR4 and LGR5

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040976

    Secretomics screen on LGR4-STF HEK293 cells. A HEK293 cell line harboring stably integrated LGR4 and the STF reporter construct were incubated with conditioned media of a library of 2859 proteins known or predicted to be secreted. Firefly luciferase values were determined for each sample and normalized to Alamar blue values of the respective samples. Media + PBS and pcDNA DEST40: Negative controls, not expressing any secreted protein; recombinant RSPO1 samples, positive controls using recombinant RSPO1 at concentrations ranging from 0.1−240 ng/ml; RSPO1 pcDNA DEST40, positive control expressing RSPO1 cDNA; Sample: Combined dataset of all secreted (known or predicted) proteins, each protein assessed from conditioned media of duplicate cDNA transfections. RSPO1-4 proteins (black squares) scored highest. Then WNT proteins (red triangles) WNT1, WNT2, WNT3A, WNT7B and WNT8A reproducibly scored above a threshold of 10 4 . 5 that marks the upper range of the bulk of datapoints, whereas WNT2B, WNT5A, WNT5B, WNT6, WNT8B, WNT9B, WNT10A, WNT10B, WNT11 and WNT16 scored below; WNT3, WNT4, WNT7A and WNT9A centered around the threshold. Each dot, triangle or square represents the luciferase value of a unicate transfected cDNA construct. Shown is an aggregate display of the primary screen and hit confirmation analysis.
    Figure Legend Snippet: Secretomics screen on LGR4-STF HEK293 cells. A HEK293 cell line harboring stably integrated LGR4 and the STF reporter construct were incubated with conditioned media of a library of 2859 proteins known or predicted to be secreted. Firefly luciferase values were determined for each sample and normalized to Alamar blue values of the respective samples. Media + PBS and pcDNA DEST40: Negative controls, not expressing any secreted protein; recombinant RSPO1 samples, positive controls using recombinant RSPO1 at concentrations ranging from 0.1−240 ng/ml; RSPO1 pcDNA DEST40, positive control expressing RSPO1 cDNA; Sample: Combined dataset of all secreted (known or predicted) proteins, each protein assessed from conditioned media of duplicate cDNA transfections. RSPO1-4 proteins (black squares) scored highest. Then WNT proteins (red triangles) WNT1, WNT2, WNT3A, WNT7B and WNT8A reproducibly scored above a threshold of 10 4 . 5 that marks the upper range of the bulk of datapoints, whereas WNT2B, WNT5A, WNT5B, WNT6, WNT8B, WNT9B, WNT10A, WNT10B, WNT11 and WNT16 scored below; WNT3, WNT4, WNT7A and WNT9A centered around the threshold. Each dot, triangle or square represents the luciferase value of a unicate transfected cDNA construct. Shown is an aggregate display of the primary screen and hit confirmation analysis.

    Techniques Used: Stable Transfection, Construct, Incubation, Luciferase, Expressing, Recombinant, Positive Control, Transfection

    4) Product Images from "Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2"

    Article Title: Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-7-20

    Murine Cβ2 cDNA encodes an active protein kinase of 47 kDa recognized by a Cβ2-specific antiserum . (A) Primers used to amplify mouse Cβ2 cDNA for insertion into pENTR/D-TOPO (Gateway system, Invitrogen). The upper 5'-end primer (upper sequence left) was designed to produce an insert with a Kozak sequence associated with the start codon (ATG). Two different 3'-end primers (lower right two sequences) were designed in order to include or exclude the stop codon (TAG). In the absence of the stop codon, the mouse Cβ2 would be expressed with a C-terminal tag, which is contained in the vector (Gateway, Invitrogen). Mouse Cβ2 cDNA was PCR amplified and subcloned into the pcDNA-DEST40 expression vector to yield pcDNA-DEST40 mCβ2. (B) HEK 293T cells (3.5 × 10 5 cells/ml) were transfected with 2μg DNA/ml of pcDNA-DEST40mCβ2 (mCβ2), mock-transfected (mock), or transfected with pEF-DEST51hCα1 (hCα1) for 24 h. Cells were lysed and analysed by SDS-PAGE and anti-pan C immunoblotting. Note that mCβ2, but not mock or hCα1 transfected cell extracts, contained an anti-C immunoreactive protein of 47 kDa. All lanes revealed a 40 kDa protein band which was most intense in cells transfected with pEF-DEST51hCα1 . (C) The same lysates were analysed for PKA-specific kinase activity in the absence (empty bars, basal) or presence (gray bars, cAMP) of 5μM cAMP, or both cAMP and PKI (black bars, cAMP/PKI). Activities were calculated relative to the activity of the mock-transfected lysate, which was set to 100 %. Bars represent mean activities of three experiments ± standard deviation. (D) The sequence of the two mCβ2 specific peptides (boxed) used to immunize rabbits to make mCβ2 specific antiserum. (E) Two rabbits were co-immunized with the two peptides and the resulting immune sera tested for immunoreactivity and specificity using cell extracts of HEK 293T cell transfected with mCβ2 (pcDNA-DEST40mCβ2) or hCα1 (pEF-DEST51hCα1). Left panel: The mCβ2 serum recognized a 47 kDa immunoreactive protein in mCβ2 but not in hCα1 transfected cell lysates. No cross-reactivity to human Cα1 could be observed. Right panel: Immunoblotting of the same lysates with a pan anti-C antibody revealed a 47 and a 40 kDa immunoreactive protein in the two extracts respectively, confirming expression of transfected constructs.
    Figure Legend Snippet: Murine Cβ2 cDNA encodes an active protein kinase of 47 kDa recognized by a Cβ2-specific antiserum . (A) Primers used to amplify mouse Cβ2 cDNA for insertion into pENTR/D-TOPO (Gateway system, Invitrogen). The upper 5'-end primer (upper sequence left) was designed to produce an insert with a Kozak sequence associated with the start codon (ATG). Two different 3'-end primers (lower right two sequences) were designed in order to include or exclude the stop codon (TAG). In the absence of the stop codon, the mouse Cβ2 would be expressed with a C-terminal tag, which is contained in the vector (Gateway, Invitrogen). Mouse Cβ2 cDNA was PCR amplified and subcloned into the pcDNA-DEST40 expression vector to yield pcDNA-DEST40 mCβ2. (B) HEK 293T cells (3.5 × 10 5 cells/ml) were transfected with 2μg DNA/ml of pcDNA-DEST40mCβ2 (mCβ2), mock-transfected (mock), or transfected with pEF-DEST51hCα1 (hCα1) for 24 h. Cells were lysed and analysed by SDS-PAGE and anti-pan C immunoblotting. Note that mCβ2, but not mock or hCα1 transfected cell extracts, contained an anti-C immunoreactive protein of 47 kDa. All lanes revealed a 40 kDa protein band which was most intense in cells transfected with pEF-DEST51hCα1 . (C) The same lysates were analysed for PKA-specific kinase activity in the absence (empty bars, basal) or presence (gray bars, cAMP) of 5μM cAMP, or both cAMP and PKI (black bars, cAMP/PKI). Activities were calculated relative to the activity of the mock-transfected lysate, which was set to 100 %. Bars represent mean activities of three experiments ± standard deviation. (D) The sequence of the two mCβ2 specific peptides (boxed) used to immunize rabbits to make mCβ2 specific antiserum. (E) Two rabbits were co-immunized with the two peptides and the resulting immune sera tested for immunoreactivity and specificity using cell extracts of HEK 293T cell transfected with mCβ2 (pcDNA-DEST40mCβ2) or hCα1 (pEF-DEST51hCα1). Left panel: The mCβ2 serum recognized a 47 kDa immunoreactive protein in mCβ2 but not in hCα1 transfected cell lysates. No cross-reactivity to human Cα1 could be observed. Right panel: Immunoblotting of the same lysates with a pan anti-C antibody revealed a 47 and a 40 kDa immunoreactive protein in the two extracts respectively, confirming expression of transfected constructs.

    Techniques Used: Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Expressing, Transfection, SDS Page, Activity Assay, Standard Deviation, Construct

    Related Articles

    Transduction:

    Article Title: Functional Linkage of Cirrhosis-Predictive Single Nucleotide Polymorphisms of Toll-like Receptor 4 to Hepatic Stellate Cell Responses
    Article Snippet: .. The destination vectors selected were pcDNA-DEST40 Gateway™ vector (Invitrogen) for the transfection of LX-2 cells, an immortalized human stellate cell line, and Plenti4/TO/V5-DEST Gateway™ vector (Invitrogen) for lentivirus-mediated transduction of hu-TLR4 cDNAs into mouse HSC lines, described as below. ..

    Clone Assay:

    Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions
    Article Snippet: .. ZIKV-NS5 cDNA was cloned into the pcDNA-DEST40 vector (Catalog No. 12274015, Thermo Fisher Scientific). pcDNA-DEST40 vector containing ZIKV-NS5 or a short flag peptide (DYKDDDDK) was transfected into HEK293T cells. .. At 48 h post transfection, cells were harvested and lysed in 1× RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mM EDTA].

    Article Title: A branched chain amino acid metabolite drives vascular transport of fat and causes insulin resistance
    Article Snippet: .. To generate VEGFB CM, a VEGFB construct was obtained from the hORFeome Database and cloned into the Gateway pcDNA-DEST40 vector (Invitrogen) and transfected into HEK293T cells. .. Fifteen hours after transfection, the cells were washed once with PBS and incubated with DMEM for 2 days before collection.

    Article Title: Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas
    Article Snippet: .. Plasmids and constructs Wild-type RET and a kinase dead version of TFG-RET (TFG-RETK270M) were cloned into Entry vectors for subsequent gateway cloning into target vectors. pENTR221 TFG-RET, pENTR221 TFG-RETK14ER21ER22E and pENTR221 TFG-RETΔ97-124 were synthesized from Thermo Fisher Scientific and cloned into expression plasmids pPHAGE C-TAP (FLAG and HA tagged), a kind from Prof. Dr. Christian Behrends, and Gateway™ pcDNA™-DEST40 Vector (His and V5-tagged) (12274015, Thermo Fisher Scientific). .. Cell culture Nthy-ori 3-1 cells (90011609, Sigma) were cultured in RPMI-1640 medium supplemented with 10% heat inactivated FBS at 37 °C in 5% CO2 .

    Article Title: Tyrosine Phosphorylation of the Human Glutathione S-Transferase P1 by Epidermal Growth Factor Receptor *
    Article Snippet: .. Cloning was performed using the Gateway technology (Invitrogen) with the pcDNA-DEST40 destination vector to allow C-terminal fusions with a six-histidine tag. .. Transient transfections were performed with FuGENE HD (Roche Applied Science) according to the manufacturer's instructions.

    Article Title: Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2
    Article Snippet: .. Expression of mouse Cβ2 For eukaryotic expression of mCβ2, the mCβ2 cDNA was cloned into the mammalian expression vector pcDNA-DEST40 (Gateway Technology System, Invitrogen), during which primers were made to introduce a Kozak sequence at the '5-end. .. In addition, two '3-end primers were used to introduce or exclude a stop codon in order to express mCβ2 with or without an N-terminal tag encoded by the expression vector itself (Invitrogen).

    Transfection:

    Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions
    Article Snippet: .. ZIKV-NS5 cDNA was cloned into the pcDNA-DEST40 vector (Catalog No. 12274015, Thermo Fisher Scientific). pcDNA-DEST40 vector containing ZIKV-NS5 or a short flag peptide (DYKDDDDK) was transfected into HEK293T cells. .. At 48 h post transfection, cells were harvested and lysed in 1× RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mM EDTA].

    Article Title: A branched chain amino acid metabolite drives vascular transport of fat and causes insulin resistance
    Article Snippet: .. To generate VEGFB CM, a VEGFB construct was obtained from the hORFeome Database and cloned into the Gateway pcDNA-DEST40 vector (Invitrogen) and transfected into HEK293T cells. .. Fifteen hours after transfection, the cells were washed once with PBS and incubated with DMEM for 2 days before collection.

    Article Title: Sphingosine kinase 1 mediates head neck squamous cell carcinoma invasion through sphingosine 1-phosphate receptor 1
    Article Snippet: .. Preparation of SphK1 plasmid and stable transfectant LR clonase enzyme (Invitrogen) was used to insert entry vector, purified pDONR223-SPHK1 (human Sphingosine Kinase-1, plasmid 23704, Addgene, Cambridge MA), into destination vector pcDNA-DEST40 Vector (12274-015, Invitrogen). .. One Shot TOP10 Chemically Competent E. coli (C4040-03, Invitrogen) was transformed with SphK1-DEST-40 plasmid DNA.

    Article Title: Functional Linkage of Cirrhosis-Predictive Single Nucleotide Polymorphisms of Toll-like Receptor 4 to Hepatic Stellate Cell Responses
    Article Snippet: .. The destination vectors selected were pcDNA-DEST40 Gateway™ vector (Invitrogen) for the transfection of LX-2 cells, an immortalized human stellate cell line, and Plenti4/TO/V5-DEST Gateway™ vector (Invitrogen) for lentivirus-mediated transduction of hu-TLR4 cDNAs into mouse HSC lines, described as below. ..

    Amplification:

    Article Title: Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2
    Article Snippet: .. To determine if the mouse Cβ2 cDNA encodes an active protein kinase, the cDNA was amplified by PCR and subcloned into pcDNA-DEST40 (Gateway Technology System, Invitrogen). .. To optimize for efficient protein expression, a Kozak sequence was introduced in the 5'-end.

    Synthesized:

    Article Title: Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas
    Article Snippet: .. Plasmids and constructs Wild-type RET and a kinase dead version of TFG-RET (TFG-RETK270M) were cloned into Entry vectors for subsequent gateway cloning into target vectors. pENTR221 TFG-RET, pENTR221 TFG-RETK14ER21ER22E and pENTR221 TFG-RETΔ97-124 were synthesized from Thermo Fisher Scientific and cloned into expression plasmids pPHAGE C-TAP (FLAG and HA tagged), a kind from Prof. Dr. Christian Behrends, and Gateway™ pcDNA™-DEST40 Vector (His and V5-tagged) (12274015, Thermo Fisher Scientific). .. Cell culture Nthy-ori 3-1 cells (90011609, Sigma) were cultured in RPMI-1640 medium supplemented with 10% heat inactivated FBS at 37 °C in 5% CO2 .

    Construct:

    Article Title: A branched chain amino acid metabolite drives vascular transport of fat and causes insulin resistance
    Article Snippet: .. To generate VEGFB CM, a VEGFB construct was obtained from the hORFeome Database and cloned into the Gateway pcDNA-DEST40 vector (Invitrogen) and transfected into HEK293T cells. .. Fifteen hours after transfection, the cells were washed once with PBS and incubated with DMEM for 2 days before collection.

    Article Title: Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas
    Article Snippet: .. Plasmids and constructs Wild-type RET and a kinase dead version of TFG-RET (TFG-RETK270M) were cloned into Entry vectors for subsequent gateway cloning into target vectors. pENTR221 TFG-RET, pENTR221 TFG-RETK14ER21ER22E and pENTR221 TFG-RETΔ97-124 were synthesized from Thermo Fisher Scientific and cloned into expression plasmids pPHAGE C-TAP (FLAG and HA tagged), a kind from Prof. Dr. Christian Behrends, and Gateway™ pcDNA™-DEST40 Vector (His and V5-tagged) (12274015, Thermo Fisher Scientific). .. Cell culture Nthy-ori 3-1 cells (90011609, Sigma) were cultured in RPMI-1640 medium supplemented with 10% heat inactivated FBS at 37 °C in 5% CO2 .

    Purification:

    Article Title: Sphingosine kinase 1 mediates head neck squamous cell carcinoma invasion through sphingosine 1-phosphate receptor 1
    Article Snippet: .. Preparation of SphK1 plasmid and stable transfectant LR clonase enzyme (Invitrogen) was used to insert entry vector, purified pDONR223-SPHK1 (human Sphingosine Kinase-1, plasmid 23704, Addgene, Cambridge MA), into destination vector pcDNA-DEST40 Vector (12274-015, Invitrogen). .. One Shot TOP10 Chemically Competent E. coli (C4040-03, Invitrogen) was transformed with SphK1-DEST-40 plasmid DNA.

    Polymerase Chain Reaction:

    Article Title: Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2
    Article Snippet: .. To determine if the mouse Cβ2 cDNA encodes an active protein kinase, the cDNA was amplified by PCR and subcloned into pcDNA-DEST40 (Gateway Technology System, Invitrogen). .. To optimize for efficient protein expression, a Kozak sequence was introduced in the 5'-end.

    Introduce:

    Article Title: Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2
    Article Snippet: .. Expression of mouse Cβ2 For eukaryotic expression of mCβ2, the mCβ2 cDNA was cloned into the mammalian expression vector pcDNA-DEST40 (Gateway Technology System, Invitrogen), during which primers were made to introduce a Kozak sequence at the '5-end. .. In addition, two '3-end primers were used to introduce or exclude a stop codon in order to express mCβ2 with or without an N-terminal tag encoded by the expression vector itself (Invitrogen).

    Expressing:

    Article Title: Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas
    Article Snippet: .. Plasmids and constructs Wild-type RET and a kinase dead version of TFG-RET (TFG-RETK270M) were cloned into Entry vectors for subsequent gateway cloning into target vectors. pENTR221 TFG-RET, pENTR221 TFG-RETK14ER21ER22E and pENTR221 TFG-RETΔ97-124 were synthesized from Thermo Fisher Scientific and cloned into expression plasmids pPHAGE C-TAP (FLAG and HA tagged), a kind from Prof. Dr. Christian Behrends, and Gateway™ pcDNA™-DEST40 Vector (His and V5-tagged) (12274015, Thermo Fisher Scientific). .. Cell culture Nthy-ori 3-1 cells (90011609, Sigma) were cultured in RPMI-1640 medium supplemented with 10% heat inactivated FBS at 37 °C in 5% CO2 .

    Article Title: Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2
    Article Snippet: .. Expression of mouse Cβ2 For eukaryotic expression of mCβ2, the mCβ2 cDNA was cloned into the mammalian expression vector pcDNA-DEST40 (Gateway Technology System, Invitrogen), during which primers were made to introduce a Kozak sequence at the '5-end. .. In addition, two '3-end primers were used to introduce or exclude a stop codon in order to express mCβ2 with or without an N-terminal tag encoded by the expression vector itself (Invitrogen).

    Sequencing:

    Article Title: Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2
    Article Snippet: .. Expression of mouse Cβ2 For eukaryotic expression of mCβ2, the mCβ2 cDNA was cloned into the mammalian expression vector pcDNA-DEST40 (Gateway Technology System, Invitrogen), during which primers were made to introduce a Kozak sequence at the '5-end. .. In addition, two '3-end primers were used to introduce or exclude a stop codon in order to express mCβ2 with or without an N-terminal tag encoded by the expression vector itself (Invitrogen).

    Plasmid Preparation:

    Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions
    Article Snippet: .. ZIKV-NS5 cDNA was cloned into the pcDNA-DEST40 vector (Catalog No. 12274015, Thermo Fisher Scientific). pcDNA-DEST40 vector containing ZIKV-NS5 or a short flag peptide (DYKDDDDK) was transfected into HEK293T cells. .. At 48 h post transfection, cells were harvested and lysed in 1× RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mM EDTA].

    Article Title: A branched chain amino acid metabolite drives vascular transport of fat and causes insulin resistance
    Article Snippet: .. To generate VEGFB CM, a VEGFB construct was obtained from the hORFeome Database and cloned into the Gateway pcDNA-DEST40 vector (Invitrogen) and transfected into HEK293T cells. .. Fifteen hours after transfection, the cells were washed once with PBS and incubated with DMEM for 2 days before collection.

    Article Title: Sphingosine kinase 1 mediates head neck squamous cell carcinoma invasion through sphingosine 1-phosphate receptor 1
    Article Snippet: .. Preparation of SphK1 plasmid and stable transfectant LR clonase enzyme (Invitrogen) was used to insert entry vector, purified pDONR223-SPHK1 (human Sphingosine Kinase-1, plasmid 23704, Addgene, Cambridge MA), into destination vector pcDNA-DEST40 Vector (12274-015, Invitrogen). .. One Shot TOP10 Chemically Competent E. coli (C4040-03, Invitrogen) was transformed with SphK1-DEST-40 plasmid DNA.

    Article Title: Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas
    Article Snippet: .. Plasmids and constructs Wild-type RET and a kinase dead version of TFG-RET (TFG-RETK270M) were cloned into Entry vectors for subsequent gateway cloning into target vectors. pENTR221 TFG-RET, pENTR221 TFG-RETK14ER21ER22E and pENTR221 TFG-RETΔ97-124 were synthesized from Thermo Fisher Scientific and cloned into expression plasmids pPHAGE C-TAP (FLAG and HA tagged), a kind from Prof. Dr. Christian Behrends, and Gateway™ pcDNA™-DEST40 Vector (His and V5-tagged) (12274015, Thermo Fisher Scientific). .. Cell culture Nthy-ori 3-1 cells (90011609, Sigma) were cultured in RPMI-1640 medium supplemented with 10% heat inactivated FBS at 37 °C in 5% CO2 .

    Article Title: Functional Linkage of Cirrhosis-Predictive Single Nucleotide Polymorphisms of Toll-like Receptor 4 to Hepatic Stellate Cell Responses
    Article Snippet: .. The destination vectors selected were pcDNA-DEST40 Gateway™ vector (Invitrogen) for the transfection of LX-2 cells, an immortalized human stellate cell line, and Plenti4/TO/V5-DEST Gateway™ vector (Invitrogen) for lentivirus-mediated transduction of hu-TLR4 cDNAs into mouse HSC lines, described as below. ..

    Article Title: Tyrosine Phosphorylation of the Human Glutathione S-Transferase P1 by Epidermal Growth Factor Receptor *
    Article Snippet: .. Cloning was performed using the Gateway technology (Invitrogen) with the pcDNA-DEST40 destination vector to allow C-terminal fusions with a six-histidine tag. .. Transient transfections were performed with FuGENE HD (Roche Applied Science) according to the manufacturer's instructions.

    Article Title: Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine C?2
    Article Snippet: .. Expression of mouse Cβ2 For eukaryotic expression of mCβ2, the mCβ2 cDNA was cloned into the mammalian expression vector pcDNA-DEST40 (Gateway Technology System, Invitrogen), during which primers were made to introduce a Kozak sequence at the '5-end. .. In addition, two '3-end primers were used to introduce or exclude a stop codon in order to express mCβ2 with or without an N-terminal tag encoded by the expression vector itself (Invitrogen).

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  • 94
    Thermo Fisher gateway pcdna dest40 vector
    Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. <t>pcDNA-DEST40</t> expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P
    Gateway Pcdna Dest40 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    90
    Thermo Fisher pcdna dest40
    Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. <t>pcDNA-DEST40</t> expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P
    Pcdna Dest40, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna dest40/product/Thermo Fisher
    Average 90 stars, based on 33 article reviews
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    pcdna dest40 - by Bioz Stars, 2020-09
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    91
    Thermo Fisher r2 pcdna dest40
    Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. <t>pcDNA-DEST40</t> expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P
    R2 Pcdna Dest40, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/r2 pcdna dest40/product/Thermo Fisher
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. pcDNA-DEST40 expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions

    doi: 10.1016/j.gpb.2018.12.010

    Figure Lengend Snippet: Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. pcDNA-DEST40 expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P

    Article Snippet: ZIKV-NS5 cDNA was cloned into the pcDNA-DEST40 vector (Catalog No. 12274015, Thermo Fisher Scientific). pcDNA-DEST40 vector containing ZIKV-NS5 or a short flag peptide (DYKDDDDK) was transfected into HEK293T cells.

    Techniques: Expressing, Transfection, Immunoprecipitation, Plasmid Preparation, SDS Page, Infection, Staining, Real-time Polymerase Chain Reaction

    Identification of protein binders for BETi JQ1 by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated JQ1 (Bio-JQ1) relative to free biotin (Bio). 12,492 ORFs were ranked according to the ratio of Bio-JQ1/Bio (from the smallest to the largest). The red line shows the cut-off value. A known protein target of JQ1, BRD2, was ranked as a top hit (red dot). B. Hit validation of JQ1 protein targets identified from the PLATO-BC assay. pET-DEST42-EWSR1/EYA3/RBM14/BD1/BD2 was transformed into the E. coli DE3 strain. V5-His6-tagged EWSR1/EYA3/RBM14/BD1/BD2 proteins were purified and subjected to protein pull-down assays for Bio-JQ1 or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. C. Interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and subjected to protein pull-down assays for biotinylated, acetylated or non-acetylated H4 (Bio-H4ac or Bio-H4) peptide. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. D. Interaction of EWSR1 with H4ac in cells. pcDNA-DEST40-EWSR1 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-His6-tagged EWSR1 using an anti-V5 mouse antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-H4ac antibody. E. JQ1 interrupts the interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and incubated with JQ1 (at 1 μM or 10 μM) or DMSO, prior to the protein pull-down assays for Bio-H4ac or free biotin (Bio). Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody or HRP-conjugated streptavidin to detect EWSR1 or H4ac respectively. BETi, bromodomain and extraterminal domain inhibitor; BRD2, bromodomain containing 2; H4ac, acetylated histone H4; EWSR1, Ewing sarcoma breakpoint region 1; EYA3, EYA transcriptional coactivator and phosphatase 3; RBM14, RNA-binding protein 14; BD1, bromodomain 1; BD2, bromodomain 2.

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions

    doi: 10.1016/j.gpb.2018.12.010

    Figure Lengend Snippet: Identification of protein binders for BETi JQ1 by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated JQ1 (Bio-JQ1) relative to free biotin (Bio). 12,492 ORFs were ranked according to the ratio of Bio-JQ1/Bio (from the smallest to the largest). The red line shows the cut-off value. A known protein target of JQ1, BRD2, was ranked as a top hit (red dot). B. Hit validation of JQ1 protein targets identified from the PLATO-BC assay. pET-DEST42-EWSR1/EYA3/RBM14/BD1/BD2 was transformed into the E. coli DE3 strain. V5-His6-tagged EWSR1/EYA3/RBM14/BD1/BD2 proteins were purified and subjected to protein pull-down assays for Bio-JQ1 or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. C. Interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and subjected to protein pull-down assays for biotinylated, acetylated or non-acetylated H4 (Bio-H4ac or Bio-H4) peptide. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. D. Interaction of EWSR1 with H4ac in cells. pcDNA-DEST40-EWSR1 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-His6-tagged EWSR1 using an anti-V5 mouse antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-H4ac antibody. E. JQ1 interrupts the interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and incubated with JQ1 (at 1 μM or 10 μM) or DMSO, prior to the protein pull-down assays for Bio-H4ac or free biotin (Bio). Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody or HRP-conjugated streptavidin to detect EWSR1 or H4ac respectively. BETi, bromodomain and extraterminal domain inhibitor; BRD2, bromodomain containing 2; H4ac, acetylated histone H4; EWSR1, Ewing sarcoma breakpoint region 1; EYA3, EYA transcriptional coactivator and phosphatase 3; RBM14, RNA-binding protein 14; BD1, bromodomain 1; BD2, bromodomain 2.

    Article Snippet: ZIKV-NS5 cDNA was cloned into the pcDNA-DEST40 vector (Catalog No. 12274015, Thermo Fisher Scientific). pcDNA-DEST40 vector containing ZIKV-NS5 or a short flag peptide (DYKDDDDK) was transfected into HEK293T cells.

    Techniques: Positron Emission Tomography, Transformation Assay, Purification, SDS Page, In Vitro, Transfection, Immunoprecipitation, Incubation, RNA Binding Assay

    Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. pcDNA-DEST40 expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions

    doi: 10.1016/j.gpb.2018.12.010

    Figure Lengend Snippet: Identification of host protein binders for the ZIKV-NS5 protein by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for V5-His6-tagged ZIKV-NS5 protein (V5-NS5) relative to a short peptide (V5-pep). 13,947 ORFs were ranked according to the ratio of V5-NS5/V5-pep (from the smallest to the largest). The red line shows the cut-off value. pcDNA-DEST40 expressing V5-tagged ZIKV-NS5 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-NS5 using an anti-V5 antibody. The precipitated protein samples were subjected to the PLATO-BC assays. B. Validation of host proteins interacting with ZIKV-NS5 using protein co-immunoprecipitation assays. pcDNA-DEST40-ZIKV-NS5 was co-transfected into HEK293T cells with the pEZY vector expressing Myc or FLAG tagged protein candidates. For majority of validations, cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged ZIKV-NS5 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using anti-V5, and anti-Myc, or FLAG antibody. For PARD3 validation, an anti-Myc antibody or a mIgG control was used for immunoprecipitation. C. Knockdown of C19orf53 and PARD3 by RNAi increases ZIKV infection. HFF-1 cells were transiently transfected with indicated siRNAs, and then infected with ZIKV. Cells were stained for expression of ZIKV capsid protein. Fluorescent images of ZIKV capsid (green) and nuclei (blue) are illustrated for HFF-1 cells treated with non-targeting siRNAs (siNTs), or siRNAs targeting known ZIKV restriction ( IFTM3 ) and dependency ( AXL ) factors, as well as PARD3 and C19orf53 . Two unique siRNAs were used for each gene. D. The infection efficiency is measured by calculating the ratio (green cells/nucleus). Relative fold of ZIKV infection in HFF-1 cells transfected with siRNAs above was normalized to those with siNTs. E. Total mRNAs were extracted from siRNA-transfected HFF-1 cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in HFF-1 cells. Relative mRNA level of ZIKV NS1 was measured in HFF −1 cells with knockdown of C19orf53 or PARD3 . F. Total mRNAs were extracted from siRNA-transfected MAGI cells, and subjected to reverse transcription and qPCR to measure knockdown efficiency of siRNA and relative NS1 mRNA level. Relative mRNA level of C19orf53 or PARD3 siRNA knockdown was normalized to that of siNT in MAGI cells. Relative mRNA level of ZIKV NS1 was measured in MAGI cells with knockdown of C19orf53 or PARD3 . Data are presented as mean ± SD ( n = 3). *, P

    Article Snippet: Tripartite motif containing 21 (TRIM21 ), Ewing sarcoma breakpoint region 1 (EWSR1 ), eyes absent homolog 3 (EYA3 ), RNA binding motif protein 14 (RMB14 ), coiled-coil domain containing 124 (CCDC124 ), par-3 family cell polarity regulator (PARD3 ), RING1 and YY1 binding protein (RYBP ), chromosome 19 open reading frame 53 (C19orf53 ), inhibitor growth of protein 2 (ING2 ) and RIO kinase 3 (RIOK3 ) were picked up from MISSION TRC3 human LentiORF library (Sigma, St. Louis, MO) and then cloned into destination vector including pcDNA-DEST40, pET-DEST42 (Catalog No. 12276010, Thermo Fisher Scientific), pEZY-Myc (Catalog No. 18701, Addgene, Watertown, MA), and pEZY-FLAG (Catalog No. 18700, Addgene).

    Techniques: Expressing, Transfection, Immunoprecipitation, Plasmid Preparation, SDS Page, Infection, Staining, Real-time Polymerase Chain Reaction

    Identification of protein binders for BETi JQ1 by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated JQ1 (Bio-JQ1) relative to free biotin (Bio). 12,492 ORFs were ranked according to the ratio of Bio-JQ1/Bio (from the smallest to the largest). The red line shows the cut-off value. A known protein target of JQ1, BRD2, was ranked as a top hit (red dot). B. Hit validation of JQ1 protein targets identified from the PLATO-BC assay. pET-DEST42-EWSR1/EYA3/RBM14/BD1/BD2 was transformed into the E. coli DE3 strain. V5-His6-tagged EWSR1/EYA3/RBM14/BD1/BD2 proteins were purified and subjected to protein pull-down assays for Bio-JQ1 or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. C. Interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and subjected to protein pull-down assays for biotinylated, acetylated or non-acetylated H4 (Bio-H4ac or Bio-H4) peptide. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. D. Interaction of EWSR1 with H4ac in cells. pcDNA-DEST40-EWSR1 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-His6-tagged EWSR1 using an anti-V5 mouse antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-H4ac antibody. E. JQ1 interrupts the interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and incubated with JQ1 (at 1 μM or 10 μM) or DMSO, prior to the protein pull-down assays for Bio-H4ac or free biotin (Bio). Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody or HRP-conjugated streptavidin to detect EWSR1 or H4ac respectively. BETi, bromodomain and extraterminal domain inhibitor; BRD2, bromodomain containing 2; H4ac, acetylated histone H4; EWSR1, Ewing sarcoma breakpoint region 1; EYA3, EYA transcriptional coactivator and phosphatase 3; RBM14, RNA-binding protein 14; BD1, bromodomain 1; BD2, bromodomain 2.

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions

    doi: 10.1016/j.gpb.2018.12.010

    Figure Lengend Snippet: Identification of protein binders for BETi JQ1 by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated JQ1 (Bio-JQ1) relative to free biotin (Bio). 12,492 ORFs were ranked according to the ratio of Bio-JQ1/Bio (from the smallest to the largest). The red line shows the cut-off value. A known protein target of JQ1, BRD2, was ranked as a top hit (red dot). B. Hit validation of JQ1 protein targets identified from the PLATO-BC assay. pET-DEST42-EWSR1/EYA3/RBM14/BD1/BD2 was transformed into the E. coli DE3 strain. V5-His6-tagged EWSR1/EYA3/RBM14/BD1/BD2 proteins were purified and subjected to protein pull-down assays for Bio-JQ1 or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. C. Interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and subjected to protein pull-down assays for biotinylated, acetylated or non-acetylated H4 (Bio-H4ac or Bio-H4) peptide. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. D. Interaction of EWSR1 with H4ac in cells. pcDNA-DEST40-EWSR1 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-His6-tagged EWSR1 using an anti-V5 mouse antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-H4ac antibody. E. JQ1 interrupts the interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and incubated with JQ1 (at 1 μM or 10 μM) or DMSO, prior to the protein pull-down assays for Bio-H4ac or free biotin (Bio). Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody or HRP-conjugated streptavidin to detect EWSR1 or H4ac respectively. BETi, bromodomain and extraterminal domain inhibitor; BRD2, bromodomain containing 2; H4ac, acetylated histone H4; EWSR1, Ewing sarcoma breakpoint region 1; EYA3, EYA transcriptional coactivator and phosphatase 3; RBM14, RNA-binding protein 14; BD1, bromodomain 1; BD2, bromodomain 2.

    Article Snippet: Tripartite motif containing 21 (TRIM21 ), Ewing sarcoma breakpoint region 1 (EWSR1 ), eyes absent homolog 3 (EYA3 ), RNA binding motif protein 14 (RMB14 ), coiled-coil domain containing 124 (CCDC124 ), par-3 family cell polarity regulator (PARD3 ), RING1 and YY1 binding protein (RYBP ), chromosome 19 open reading frame 53 (C19orf53 ), inhibitor growth of protein 2 (ING2 ) and RIO kinase 3 (RIOK3 ) were picked up from MISSION TRC3 human LentiORF library (Sigma, St. Louis, MO) and then cloned into destination vector including pcDNA-DEST40, pET-DEST42 (Catalog No. 12276010, Thermo Fisher Scientific), pEZY-Myc (Catalog No. 18701, Addgene, Watertown, MA), and pEZY-FLAG (Catalog No. 18700, Addgene).

    Techniques: Positron Emission Tomography, Transformation Assay, Purification, SDS Page, In Vitro, Transfection, Immunoprecipitation, Incubation, RNA Binding Assay