pcdna3 1 mammalian expression vector  (Thermo Fisher)


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    Name:
    pcDNA 3 1 Mammalian Expression Vector
    Description:
    This pcDNA 3 1 vector is designed for high level constitutive expression in a variety of mammalian cell lines It contains a Geneticin selectable marker and a forward orientation multiple cloning site The pcDNA 3 1 Expression Vector FamilyThree untagged versions of pcDNA 3 1 available separately each with a different selectable marker Geneticin Zeocin or Hygromycin are for use alone or in co transfections All three vectors offer the following features • Cytomegalovirus CMV enhancer promoter for high level expression• Large multiple cloning site in either forward or reverse orientations• Bovine Growth Hormone BGH polyadenylation signal and transcription termination sequence for enhanced mRNA stability• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen i e COS 1 and COS 7 • Ampicillin resistance gene and pUC origin for selection and maintenance in E coli
    Catalog Number:
    v79020
    Price:
    None
    Applications:
    Constitutive Expression|Mammalian Expression|Protein Biology|Protein Expression
    Category:
    DNA Vectors Clones Purified Nucleic Acids Libraries
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    Structured Review

    Thermo Fisher pcdna3 1 mammalian expression vector
    HBx downregulates the expression of TNFRSF10B in HBV-infected cells. (A) TNFRSF10B expression levels in HepG-X and HepG-M cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (B) TNFRSF10B expression at the indicated time points after transfection with the control or HBx plasmid in HepG2 cells was analyzed by immunoblot assay. A representative immunoblot and quantification of the TNFRSF10B signal are shown. (C) TNFRSF10B expression levels in L02-X and L02-M cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (D) TNFRSF10B expression at the indicated time points after transfection of L02 cells with the control or HBx plasmid was analyzed by immunoblot assay. Quantification of the TNFRSF10B signal is shown. (E) Expression levels of TNFRSF10B in HepG2 and HepG2.2.15 cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (F) TNFRSF10B expression at the indicated time points after transfection in HepG2 cells with the control or HBV1.2mer plasmid was analyzed by immunoblot assay. A representative immunoblot and quantification of the TNFRSF10B signal are shown. (G) Representative immunocytochemical images of TNFRSF10B expression in HepG2 cells transiently transfected with the HBx plasmid. Cells transfected with the HBx-Flag or <t>pcDNA3.1</t> vector were stained with anti-Flag (red) and anti-TNFRSF10B (green) antibodies. Among the HBx-positive cells, the percentage of cells showing reduced TNFRSF10B expression compared to control cells is indicated. (H) TNFRSF10B expression levels on the surface of HBx-expressing cells, HepG2.2.15 cells, and the corresponding control cells were analyzed by flow cytometry. Relative TNFRSF10B expression on the cell surface was calculated as the percentage of mean fluorescence intensity (MFI) as described in the Materials and Methods. All data are mean ± SD of 3 independent experiments. P -values were obtained by Student t test (* P
    This pcDNA 3 1 vector is designed for high level constitutive expression in a variety of mammalian cell lines It contains a Geneticin selectable marker and a forward orientation multiple cloning site The pcDNA 3 1 Expression Vector FamilyThree untagged versions of pcDNA 3 1 available separately each with a different selectable marker Geneticin Zeocin or Hygromycin are for use alone or in co transfections All three vectors offer the following features • Cytomegalovirus CMV enhancer promoter for high level expression• Large multiple cloning site in either forward or reverse orientations• Bovine Growth Hormone BGH polyadenylation signal and transcription termination sequence for enhanced mRNA stability• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen i e COS 1 and COS 7 • Ampicillin resistance gene and pUC origin for selection and maintenance in E coli
    https://www.bioz.com/result/pcdna3 1 mammalian expression vector/product/Thermo Fisher
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    Images

    1) Product Images from "Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response"

    Article Title: Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

    Journal: Autophagy

    doi: 10.1080/15548627.2016.1239002

    HBx downregulates the expression of TNFRSF10B in HBV-infected cells. (A) TNFRSF10B expression levels in HepG-X and HepG-M cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (B) TNFRSF10B expression at the indicated time points after transfection with the control or HBx plasmid in HepG2 cells was analyzed by immunoblot assay. A representative immunoblot and quantification of the TNFRSF10B signal are shown. (C) TNFRSF10B expression levels in L02-X and L02-M cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (D) TNFRSF10B expression at the indicated time points after transfection of L02 cells with the control or HBx plasmid was analyzed by immunoblot assay. Quantification of the TNFRSF10B signal is shown. (E) Expression levels of TNFRSF10B in HepG2 and HepG2.2.15 cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (F) TNFRSF10B expression at the indicated time points after transfection in HepG2 cells with the control or HBV1.2mer plasmid was analyzed by immunoblot assay. A representative immunoblot and quantification of the TNFRSF10B signal are shown. (G) Representative immunocytochemical images of TNFRSF10B expression in HepG2 cells transiently transfected with the HBx plasmid. Cells transfected with the HBx-Flag or pcDNA3.1 vector were stained with anti-Flag (red) and anti-TNFRSF10B (green) antibodies. Among the HBx-positive cells, the percentage of cells showing reduced TNFRSF10B expression compared to control cells is indicated. (H) TNFRSF10B expression levels on the surface of HBx-expressing cells, HepG2.2.15 cells, and the corresponding control cells were analyzed by flow cytometry. Relative TNFRSF10B expression on the cell surface was calculated as the percentage of mean fluorescence intensity (MFI) as described in the Materials and Methods. All data are mean ± SD of 3 independent experiments. P -values were obtained by Student t test (* P
    Figure Legend Snippet: HBx downregulates the expression of TNFRSF10B in HBV-infected cells. (A) TNFRSF10B expression levels in HepG-X and HepG-M cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (B) TNFRSF10B expression at the indicated time points after transfection with the control or HBx plasmid in HepG2 cells was analyzed by immunoblot assay. A representative immunoblot and quantification of the TNFRSF10B signal are shown. (C) TNFRSF10B expression levels in L02-X and L02-M cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (D) TNFRSF10B expression at the indicated time points after transfection of L02 cells with the control or HBx plasmid was analyzed by immunoblot assay. Quantification of the TNFRSF10B signal is shown. (E) Expression levels of TNFRSF10B in HepG2 and HepG2.2.15 cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (F) TNFRSF10B expression at the indicated time points after transfection in HepG2 cells with the control or HBV1.2mer plasmid was analyzed by immunoblot assay. A representative immunoblot and quantification of the TNFRSF10B signal are shown. (G) Representative immunocytochemical images of TNFRSF10B expression in HepG2 cells transiently transfected with the HBx plasmid. Cells transfected with the HBx-Flag or pcDNA3.1 vector were stained with anti-Flag (red) and anti-TNFRSF10B (green) antibodies. Among the HBx-positive cells, the percentage of cells showing reduced TNFRSF10B expression compared to control cells is indicated. (H) TNFRSF10B expression levels on the surface of HBx-expressing cells, HepG2.2.15 cells, and the corresponding control cells were analyzed by flow cytometry. Relative TNFRSF10B expression on the cell surface was calculated as the percentage of mean fluorescence intensity (MFI) as described in the Materials and Methods. All data are mean ± SD of 3 independent experiments. P -values were obtained by Student t test (* P

    Techniques Used: Expressing, Infection, Quantitative RT-PCR, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Fluorescence

    2) Product Images from "Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor"

    Article Title: Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04432-0

    Nb5 as a control-switch for GPCR-mediated Gβγ signaling. a Schematic diagram of the GPCR-mediated Gβγ signaling network that controls various cellular functions. b Parental CHO-APJ cells, CHO-APJ cells transfected with pcDNA3.1 (+) empty vector, transfected CHO-APJ-Nb5 and CHO-APJ-Nb17 cells were treated with 1 μM apelin over 0–5 min to investigate the effect of Nb5 on the phosphorylation of ERK1/2 and AKT. Nb5 significantly decreased the phosphorylation of both ERK1/2 and AKT as compared to the Nb17 control (compare lanes marked with asterisks). Full blots are reported in Supplementary Figure 4b . c No significant effect of Nb17 transfection (purple) was observed on the phosphorylation of either ERK1/2 or AKT as compared to parental CHO-APJ cells (black) and CHO-APJ cells transfected with pcDNA3.1 (+) empty vector (red). Results are expressed as the mean ± SEM, n = 3 replicates, * P
    Figure Legend Snippet: Nb5 as a control-switch for GPCR-mediated Gβγ signaling. a Schematic diagram of the GPCR-mediated Gβγ signaling network that controls various cellular functions. b Parental CHO-APJ cells, CHO-APJ cells transfected with pcDNA3.1 (+) empty vector, transfected CHO-APJ-Nb5 and CHO-APJ-Nb17 cells were treated with 1 μM apelin over 0–5 min to investigate the effect of Nb5 on the phosphorylation of ERK1/2 and AKT. Nb5 significantly decreased the phosphorylation of both ERK1/2 and AKT as compared to the Nb17 control (compare lanes marked with asterisks). Full blots are reported in Supplementary Figure 4b . c No significant effect of Nb17 transfection (purple) was observed on the phosphorylation of either ERK1/2 or AKT as compared to parental CHO-APJ cells (black) and CHO-APJ cells transfected with pcDNA3.1 (+) empty vector (red). Results are expressed as the mean ± SEM, n = 3 replicates, * P

    Techniques Used: Transfection, Plasmid Preparation

    3) Product Images from "TWEAK Negatively Regulates Human Dicer"

    Article Title: TWEAK Negatively Regulates Human Dicer

    Journal: Non-Coding RNA

    doi: 10.3390/ncrna2040012

    TWEAK dose-dependently reduces pre-microRNA conversion into mature microRNA by Dicer. ( a , b ) Cleared lysates (50 µg of proteins) prepared from HEK 293 cells, transfected with increasing amounts of pcDNA3.1-Flag-TWEAK vector (0–20 µg of DNA), were incubated with 5′ 32 P-labeled human pre-miR-223 (miRBase acc. no. MI0000300) or pre-let-7c (miRBase acc. no. MI0018703). ( a ) The 32 P-labeled pre-microRNA substrates and cleavage products of Dicer were revealed by denaturing polyacrylamide gel electrophoresis (PAGE)/autoradiography; ( b ) The ≈20-nt bands corresponding to the mature microRNA products were analyzed by densitometry using the ImageJ ® software; ( c ) The Dicer protein level in the HEK 293 cell lysates was analyzed by immunoblotting using anti-Dicer antibody, with anti-tubulin as a reference.
    Figure Legend Snippet: TWEAK dose-dependently reduces pre-microRNA conversion into mature microRNA by Dicer. ( a , b ) Cleared lysates (50 µg of proteins) prepared from HEK 293 cells, transfected with increasing amounts of pcDNA3.1-Flag-TWEAK vector (0–20 µg of DNA), were incubated with 5′ 32 P-labeled human pre-miR-223 (miRBase acc. no. MI0000300) or pre-let-7c (miRBase acc. no. MI0018703). ( a ) The 32 P-labeled pre-microRNA substrates and cleavage products of Dicer were revealed by denaturing polyacrylamide gel electrophoresis (PAGE)/autoradiography; ( b ) The ≈20-nt bands corresponding to the mature microRNA products were analyzed by densitometry using the ImageJ ® software; ( c ) The Dicer protein level in the HEK 293 cell lysates was analyzed by immunoblotting using anti-Dicer antibody, with anti-tubulin as a reference.

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Software

    TWEAK impairs microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. HEK 293 cells expressing Flag-TWEAK (or transfected with an empty pcDNA3.1-Flag vector; Flag) were transfected with the silencing inducer pre-miR-328a (or a negative pre-microRNA control targeting a deleted region in Rluc ; shNEG), and a Rluc reporter gene [ 16 ] coupled with three copies of a natural miR-328a binding site in the 3′ untranslated region (UTR) of the Rluc reporter gene. Cells were harvested 18 h later for the successive measurements of Rluc and Fluc activities ( n = 3 experiments, in duplicate). * p
    Figure Legend Snippet: TWEAK impairs microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. HEK 293 cells expressing Flag-TWEAK (or transfected with an empty pcDNA3.1-Flag vector; Flag) were transfected with the silencing inducer pre-miR-328a (or a negative pre-microRNA control targeting a deleted region in Rluc ; shNEG), and a Rluc reporter gene [ 16 ] coupled with three copies of a natural miR-328a binding site in the 3′ untranslated region (UTR) of the Rluc reporter gene. Cells were harvested 18 h later for the successive measurements of Rluc and Fluc activities ( n = 3 experiments, in duplicate). * p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Binding Assay

    Dicer and TWEAK proteins form a complex likely independent of RNA in HEK 293 cells. HEK 293 cells were transiently transfected with pcDNA3.1-Flag-TWEAK vector, or an empty pcDNA3.1-Flag vector (Flag), prior to Flag-TWEAK protein immunoprecipitation (IP) from untreated lysate (lanes 1–3 and 6–8) or lysate treated with RNases A/T1/V1 (+RNases; lanes 4–5 and 9–10). ( a ) The presence of Flag-TWEAK and Dicer proteins in the lysates (Input) and the IPs was verified by immunoblot (IB) analysis using anti-Flag and anti-Dicer antibodies. The two bands immunoreactive to the anti-Flag antibody likely correspond to different posttranslational forms of the Flag-tagged TWEAK protein. The band across lanes 6 to 10 of the IB anti-Flag panel likely corresponds to the light chain of mouse IgGs; ( b ) RNA degradation upon treatment with RNases A/T1/V1 was confirmed by agarose gel electrophoresis and ethidium bromide staining.
    Figure Legend Snippet: Dicer and TWEAK proteins form a complex likely independent of RNA in HEK 293 cells. HEK 293 cells were transiently transfected with pcDNA3.1-Flag-TWEAK vector, or an empty pcDNA3.1-Flag vector (Flag), prior to Flag-TWEAK protein immunoprecipitation (IP) from untreated lysate (lanes 1–3 and 6–8) or lysate treated with RNases A/T1/V1 (+RNases; lanes 4–5 and 9–10). ( a ) The presence of Flag-TWEAK and Dicer proteins in the lysates (Input) and the IPs was verified by immunoblot (IB) analysis using anti-Flag and anti-Dicer antibodies. The two bands immunoreactive to the anti-Flag antibody likely correspond to different posttranslational forms of the Flag-tagged TWEAK protein. The band across lanes 6 to 10 of the IB anti-Flag panel likely corresponds to the light chain of mouse IgGs; ( b ) RNA degradation upon treatment with RNases A/T1/V1 was confirmed by agarose gel electrophoresis and ethidium bromide staining.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Agarose Gel Electrophoresis, Staining

    4) Product Images from "TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis"

    Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2017.23

    Validation of TAK1 as a molecular target of resveratrol and identification of key residues in TAK1. ( a ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between TAK1 and resveratrol (Res) in alveolar macrophage NR8383 (left) and lung fibroblasts WI-38 cells (right). TAK1 in the immunoprecipitate and lysate was examined by western blotting (upper: representative images; bottom: relative bands intensity). ( b ) Reference standard of resveratrol (left upper) and immunoprecipitation using anti-TAK1 antibody to examine the interaction between resveratrol and TAK1 in NR8383 (left bottom) and WI-38 cells (right). Resveratrol in the immunoprecipitate was examined by LC-MS/MS. There were two peaks for trans - and cis -resveratrol according to the reference standard. ( c ) Close-up view showing key residues on TAK1 in two predicted binding conformations between TAK1 and resveratrol in molecule docking. ( d ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R) and resveratrol in NR8383 (left) and WI-38 cells (right). The cells were transfected with pcDNA3.1-based expression vectors for Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R). Flag-TAK1 or Flag-TAK1 mutants in immunoprecipitates and lysates was determined by western blotting (upper: representative images; bottom: relative bands intensity). ( e ) Immunoprecipitation using anti-Flag antibody to examine the interaction between resveratrol and Flag-TAK1 wild-type or Flag-TAK1 mutants in NR8383 cells (upper) and WI-38 cells (bottom). Resveratrol in immunoprecipitate was examined by LC-MS/MS. Experiments were performed at least three times. Res, resveratrol; ND, not detectable.
    Figure Legend Snippet: Validation of TAK1 as a molecular target of resveratrol and identification of key residues in TAK1. ( a ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between TAK1 and resveratrol (Res) in alveolar macrophage NR8383 (left) and lung fibroblasts WI-38 cells (right). TAK1 in the immunoprecipitate and lysate was examined by western blotting (upper: representative images; bottom: relative bands intensity). ( b ) Reference standard of resveratrol (left upper) and immunoprecipitation using anti-TAK1 antibody to examine the interaction between resveratrol and TAK1 in NR8383 (left bottom) and WI-38 cells (right). Resveratrol in the immunoprecipitate was examined by LC-MS/MS. There were two peaks for trans - and cis -resveratrol according to the reference standard. ( c ) Close-up view showing key residues on TAK1 in two predicted binding conformations between TAK1 and resveratrol in molecule docking. ( d ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R) and resveratrol in NR8383 (left) and WI-38 cells (right). The cells were transfected with pcDNA3.1-based expression vectors for Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R). Flag-TAK1 or Flag-TAK1 mutants in immunoprecipitates and lysates was determined by western blotting (upper: representative images; bottom: relative bands intensity). ( e ) Immunoprecipitation using anti-Flag antibody to examine the interaction between resveratrol and Flag-TAK1 wild-type or Flag-TAK1 mutants in NR8383 cells (upper) and WI-38 cells (bottom). Resveratrol in immunoprecipitate was examined by LC-MS/MS. Experiments were performed at least three times. Res, resveratrol; ND, not detectable.

    Techniques Used: Immunoprecipitation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Binding Assay, Transfection, Expressing

    5) Product Images from "Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope"

    Article Title: Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope

    Journal: Viruses

    doi: 10.3390/v8030073

    Interaction between RISP with HCMV pUL50 in human cells. ( a , b ) Coimmunoprecipitation (CoIP) assay. Human 293T cells were transiently cotransfected with constructs coding for FLAG-tagged RISP and HA-tagged HCMV proteins pUL50(1–358) (ratio 1:1 and 1:3, lanes 3 and 4, respectively), pUL97 (lane 5) or pUL53 (lane 6), or with an empty vector (pcDNA3.1, lane 2) as control. Coexpression of HA-tagged pUL50(1–358) and FLAG-tagged pUL53 served as CoIP control (lane 7); RFP expression served as transfection control (lane 1). At 2 days post-transfection, cells were lysed, and HA-tagged proteins were immunoprecipitated using mouse monoclonal antibody mAb-HA. CoIP samples ( a ) and lysate controls (input) ( b ) taken prior to the addition of the CoIP antibody were subjected to standard Western blot analysis using tag-specific antibodies as indicated. Ig-HC, cross-reactive band for immunoglobulin heavy chain.
    Figure Legend Snippet: Interaction between RISP with HCMV pUL50 in human cells. ( a , b ) Coimmunoprecipitation (CoIP) assay. Human 293T cells were transiently cotransfected with constructs coding for FLAG-tagged RISP and HA-tagged HCMV proteins pUL50(1–358) (ratio 1:1 and 1:3, lanes 3 and 4, respectively), pUL97 (lane 5) or pUL53 (lane 6), or with an empty vector (pcDNA3.1, lane 2) as control. Coexpression of HA-tagged pUL50(1–358) and FLAG-tagged pUL53 served as CoIP control (lane 7); RFP expression served as transfection control (lane 1). At 2 days post-transfection, cells were lysed, and HA-tagged proteins were immunoprecipitated using mouse monoclonal antibody mAb-HA. CoIP samples ( a ) and lysate controls (input) ( b ) taken prior to the addition of the CoIP antibody were subjected to standard Western blot analysis using tag-specific antibodies as indicated. Ig-HC, cross-reactive band for immunoglobulin heavy chain.

    Techniques Used: Co-Immunoprecipitation Assay, Construct, Plasmid Preparation, Expressing, Transfection, Immunoprecipitation, Western Blot

    6) Product Images from "Estradiol-Estrogen Receptor α Mediates the Expression of the CXXC5 Gene through the Estrogen Response Element-Dependent Signaling Pathway"

    Article Title: Estradiol-Estrogen Receptor α Mediates the Expression of the CXXC5 Gene through the Estrogen Response Element-Dependent Signaling Pathway

    Journal: Scientific Reports

    doi: 10.1038/srep37808

    Intracellular Localization of CXXC5. MCF7 cells grown on coverslips in 12-well culture plates with medium containing FBS for 48 h were un-transfected (UT) or transfected with pcDNA3.1(−) bearing the Flag-CXXC5 (F-C5) cDNA. Thirty six hour after, cells were fixed with 2% paraformaldehyde in PBS and permeabilized with 0.4% Triton-X100 in PBS. For the detection of the endogenous CXXC5 protein in un-transfected cells, cells were blocked with 10% normal goat serum (NGS) followed by an incubation with ab106533 in PBS containing 2% NGS. Cells were then incubated with an Alexa Fluor ® -488 (green channel) conjugated goat anti-rabbit secondary antibody in PBS containing 2% NGS to detect endogenous CXXC5. For the detection of Flag-CXXC5 protein in transfected cells, following a block with 10% bovine serum albumin (BSA) in PBS, cells were incubated with the Flag-M2 antibody in PBS containing 3% BSA. Cells were then incubated with an Alexa Fluor ® -488 (green channel) conjugated goat anti-mouse secondary antibody in PBS containing 3% BSA. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (blue channel). Merge images are indicated. A representative image from two independent experiments is shown. Scale bar is 5 μm.
    Figure Legend Snippet: Intracellular Localization of CXXC5. MCF7 cells grown on coverslips in 12-well culture plates with medium containing FBS for 48 h were un-transfected (UT) or transfected with pcDNA3.1(−) bearing the Flag-CXXC5 (F-C5) cDNA. Thirty six hour after, cells were fixed with 2% paraformaldehyde in PBS and permeabilized with 0.4% Triton-X100 in PBS. For the detection of the endogenous CXXC5 protein in un-transfected cells, cells were blocked with 10% normal goat serum (NGS) followed by an incubation with ab106533 in PBS containing 2% NGS. Cells were then incubated with an Alexa Fluor ® -488 (green channel) conjugated goat anti-rabbit secondary antibody in PBS containing 2% NGS to detect endogenous CXXC5. For the detection of Flag-CXXC5 protein in transfected cells, following a block with 10% bovine serum albumin (BSA) in PBS, cells were incubated with the Flag-M2 antibody in PBS containing 3% BSA. Cells were then incubated with an Alexa Fluor ® -488 (green channel) conjugated goat anti-mouse secondary antibody in PBS containing 3% BSA. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (blue channel). Merge images are indicated. A representative image from two independent experiments is shown. Scale bar is 5 μm.

    Techniques Used: Transfection, Next-Generation Sequencing, Incubation, Blocking Assay, Staining

    Detection of CXXC5 protein in MCF7 cells. ( a ) MCF7 cells were un-transfected (UT) or transfected with pcDNA3.1 (−) bearing WT-CXXC5 (WT-C5) or Flag-CXXC5 (F-C5) cDNA for 24 h. Cells were then subjected to nuclear protein extraction, SDS 10%-PAGE and WB using a CXXC5-specific antibody, ab106533. Star in the un-transfected (UT) lane denotes the putative endogenous CXXC5 protein, while arrows indicate the overexpressed WT-CXXC5 or Flag-CXXC. Molecular weight marker is in kDa. A representative image from two independent experiments is shown. It should be noted that we used 100 μg nuclear extracts of UT cells, while 25 μg of nuclear extracts of WT-C5 or F-C5 cells were used to prevent overshadowing effects of the overexpressed CXXC5 ( Supplemental Data, Fig. 1 ). ( b ) MCF7 cells were transfected without (un-transfected, UT) or with AllStars negative control siRNA (NC), siRNA#2, #7, #9 or #10. 24 h later, cells were subjected to total RNA extractions and RT-qPCR using CXXC5 specific-primers. CXXC5 transcript levels in siRNA transfected cells were compared to levels in un-transfected cells, which was set to 1. Results are the mean ± SD of three biological repeats with three technical replicates. Asterisk (*) denotes significant change. ( c ) MCF7 cells were transfected for 24 h without (un-transfected, UT) or with AllStars (NC), siRNA#2, #7, #9 or #10. We also transfected cells with pcDNA3.1 bearing none (Vector, V) or WT-CXXC5 cDNA as control. 100 μg nuclear protein extracts, with the exception of F-C5 which was 25 μg to prevent the shadowing effect of the overexpressed protein on the endogenous protein, were subjected to WB using ab106533. Star denotes the endogenous CXXC5, while the arrow indicates the overexpressed Flag-CXXC5. A representative image from two independent experiments is shown. ( d ) Effects of ER ligands on endogenous CXXC5. MCF7 cells grown in medium containing CD-FBS for 48 h were treated without (EtOH, 0.01%) or with 10 −9 M E2 and/or 10 −7 M ICI for 24 hours. Nuclear extracts were subjected to WB using ab106533 or an HDAC1 antibody. A representative image from two independent experiments is shown. Changes in protein levels were quantified with ImageJ image processing program. Asterisk (*) denotes significant change.
    Figure Legend Snippet: Detection of CXXC5 protein in MCF7 cells. ( a ) MCF7 cells were un-transfected (UT) or transfected with pcDNA3.1 (−) bearing WT-CXXC5 (WT-C5) or Flag-CXXC5 (F-C5) cDNA for 24 h. Cells were then subjected to nuclear protein extraction, SDS 10%-PAGE and WB using a CXXC5-specific antibody, ab106533. Star in the un-transfected (UT) lane denotes the putative endogenous CXXC5 protein, while arrows indicate the overexpressed WT-CXXC5 or Flag-CXXC. Molecular weight marker is in kDa. A representative image from two independent experiments is shown. It should be noted that we used 100 μg nuclear extracts of UT cells, while 25 μg of nuclear extracts of WT-C5 or F-C5 cells were used to prevent overshadowing effects of the overexpressed CXXC5 ( Supplemental Data, Fig. 1 ). ( b ) MCF7 cells were transfected without (un-transfected, UT) or with AllStars negative control siRNA (NC), siRNA#2, #7, #9 or #10. 24 h later, cells were subjected to total RNA extractions and RT-qPCR using CXXC5 specific-primers. CXXC5 transcript levels in siRNA transfected cells were compared to levels in un-transfected cells, which was set to 1. Results are the mean ± SD of three biological repeats with three technical replicates. Asterisk (*) denotes significant change. ( c ) MCF7 cells were transfected for 24 h without (un-transfected, UT) or with AllStars (NC), siRNA#2, #7, #9 or #10. We also transfected cells with pcDNA3.1 bearing none (Vector, V) or WT-CXXC5 cDNA as control. 100 μg nuclear protein extracts, with the exception of F-C5 which was 25 μg to prevent the shadowing effect of the overexpressed protein on the endogenous protein, were subjected to WB using ab106533. Star denotes the endogenous CXXC5, while the arrow indicates the overexpressed Flag-CXXC5. A representative image from two independent experiments is shown. ( d ) Effects of ER ligands on endogenous CXXC5. MCF7 cells grown in medium containing CD-FBS for 48 h were treated without (EtOH, 0.01%) or with 10 −9 M E2 and/or 10 −7 M ICI for 24 hours. Nuclear extracts were subjected to WB using ab106533 or an HDAC1 antibody. A representative image from two independent experiments is shown. Changes in protein levels were quantified with ImageJ image processing program. Asterisk (*) denotes significant change.

    Techniques Used: Transfection, Protein Extraction, Polyacrylamide Gel Electrophoresis, Western Blot, Molecular Weight, Marker, Negative Control, Quantitative RT-PCR, Plasmid Preparation

    Electrophoretic mobility Shift Assay (EMSA). ( a ) The upper and lower oligomer sequences, which surround (parenthesis) the consensus-ERE (Con-ERE) or CXXC5-ERE test sequence, are biotinylated at 5′-ends. Underlined A residue in CXXC5-ERE indicates the variation from the consensus. ( b,c ) Cell extracts (CE; 10 μg) of MDAMB231 cells transfected with pcDNA3.1(−) bearing none (Vector, V) or the Flag-ERα cDNA were subjected to EMSA using biotinylated DNA (40 fmol) with (+) or without (−) the Flag-M2 antibody (Flag-M2) in the absence (−) or presence (+) of cold competitor at indicated amounts. ERα-ERE denotes the protein-bound biotinylated ERE. ERE indicates the unbound (free) biotinylated ERE. A representative result from three independent determinations is shown.
    Figure Legend Snippet: Electrophoretic mobility Shift Assay (EMSA). ( a ) The upper and lower oligomer sequences, which surround (parenthesis) the consensus-ERE (Con-ERE) or CXXC5-ERE test sequence, are biotinylated at 5′-ends. Underlined A residue in CXXC5-ERE indicates the variation from the consensus. ( b,c ) Cell extracts (CE; 10 μg) of MDAMB231 cells transfected with pcDNA3.1(−) bearing none (Vector, V) or the Flag-ERα cDNA were subjected to EMSA using biotinylated DNA (40 fmol) with (+) or without (−) the Flag-M2 antibody (Flag-M2) in the absence (−) or presence (+) of cold competitor at indicated amounts. ERα-ERE denotes the protein-bound biotinylated ERE. ERE indicates the unbound (free) biotinylated ERE. A representative result from three independent determinations is shown.

    Techniques Used: Electrophoretic Mobility Shift Assay, Sequencing, Transfection, Plasmid Preparation

    Related Articles

    Diagnostic Assay:

    Article Title: HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924
    Article Snippet: The pcDNA 3.1 mammalian expression vector was purchased from Invitrogen (Carlsbad, CA). pcDNA 3.1 vector containing wild-type EGFR was a gift from C. David James. pcDNA 3.1 vector containing wild-type HER2 was a gift from Tai Wong. .. The appropriate vector clones were then verified by diagnostic (Hind III/Xba I) digesting small scale mini preparations (Promega) and then verified by DNA sequencing.

    Clone Assay:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: The ATG5-K130R mutant replacing Lys130 with Arg was generated by PCR and cloned into the Xhol and Xbal sites of pcDNA3.1-HA. .. The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020).

    Article Title: HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924
    Article Snippet: The pcDNA 3.1 mammalian expression vector was purchased from Invitrogen (Carlsbad, CA). pcDNA 3.1 vector containing wild-type EGFR was a gift from C. David James. pcDNA 3.1 vector containing wild-type HER2 was a gift from Tai Wong. .. The appropriate vector clones were then verified by diagnostic (Hind III/Xba I) digesting small scale mini preparations (Promega) and then verified by DNA sequencing.

    Article Title: FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes
    Article Snippet: .. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures. .. Recombinant human FAM3B protein assays In order to verify the effects of exogenous FAM3B on cell viability 1 × 106 DU145 cells were seeded onto 2 cm diameter dishes and treated with recombinant human FAM3B protein (rhFAM3B) obtained in bacterial host.

    Article Title: Mechanism of RPE Cell Death in ?-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux
    Article Snippet: Construction of αA and αB-crystallin cDNAs Full-length αA and αB-crystallin cDNAs were amplified from human fetal lens and fetal RPE, respectively, and cloned into a mammalian expression vector. .. The PCR products were digested with EcoR1 and Zho1, and then ligated into pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, USA) having a neomycin resistance gene for selection.

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators
    Article Snippet: Briefly, the corresponding TALE DNA-binding domain was constructed and cloned into pTAL destination vector using Golden Gate cloning method. .. The TALE DNA-binding domain was excised from pTAL using EcoRI and Bam H1 and subsequently subcloned into pcDNA-3.1 mammalian expression vector containing a CMV promoter (Invitrogen).

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. For plasmids expressing Venus-tagged PER1, PER2, CRY1, CRY2 (PER1-Venus, PER2-Venus, CRY1-Venus, CRY2-Venus) or BiFC plasmids expressing Venus N terminal (VN) or C-terminal (VC)-tagged PER1, PER2, CRY1, and CRY2 (PER1-VN, PER2-VN, CRY1-VC, CRY2-VC), full-length DNA fragment of the each of genes was subcloned into pCMV-Venus, pCMV-VN, or pCMV-VC using a restriction-free cloning method as described previously ( ; ).

    Article Title: Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus
    Article Snippet: .. The non-structural NS3 gene of the dengue virus local isolate was cloned into pcDNA 3.1 Mammalian Expression Vector (Invitrogen, USA) by ligation of the amplified NS3 gene product into the digested vector and transformed into the bacterial cells. .. The NS3 mammalian expression vector constructs are shown in .

    Article Title: Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation
    Article Snippet: Sox2 knockdown in low-passage 10% FBS cells was achieved by oligonucleotides targeting human SOX2-coding or nonsilencing control sequences cloned into BLOCK-iT Pol II miR RNAi expression vectors (Life Technologies) before cell transfection with Lipofectamine 2000 (Life Technologies). .. Sox2 ectopic expression was achieved by subcloning Sox2 cDNA from pCMV6-XL5- (OriGene) into pcDNA 3.1 mammalian expression vector (Invitrogen), under control of constitutive CMV promoter; the empty vector was used as control.

    Amplification:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: The full-length ATG5 gene was amplified from genomic DNA of the HK-2 cell line by PCR and subcloned into the pcDNA3.1 vector (Invitrogen,V79020). .. The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020).

    Article Title: HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924
    Article Snippet: The pcDNA 3.1 mammalian expression vector was purchased from Invitrogen (Carlsbad, CA). pcDNA 3.1 vector containing wild-type EGFR was a gift from C. David James. pcDNA 3.1 vector containing wild-type HER2 was a gift from Tai Wong. .. The vectors were amplified transforming chemically competent E. coli and selecting on LB + ampicillin culture plates.

    Article Title: FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes
    Article Snippet: Cloning of FAM3B cDNA into lentiviral and plasmidial vectors Human FAM3B was amplified by RT-PCR from human pancreatic islets mRNA. .. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures.

    Article Title: Mechanism of RPE Cell Death in ?-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux
    Article Snippet: Briefly, full-length α-crystallin cDNA (αA and αB crystallin) were amplified using the primer sequences ( ). .. The PCR products were digested with EcoR1 and Zho1, and then ligated into pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, USA) having a neomycin resistance gene for selection.

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators
    Article Snippet: The TALE DNA-binding domain was excised from pTAL using EcoRI and Bam H1 and subsequently subcloned into pcDNA-3.1 mammalian expression vector containing a CMV promoter (Invitrogen). .. The VP64 DNA fragment was PCR amplified from plasmid pLenti-EF1a-Backbone (NI) obtained from addgene ( http://www.addgene.org/27962 ) and a SV40 nuclear localization signal added to N-terminus of VP64 using primers NLS_VP64 forward: 5′CCGCGGAGCCCCAAGAAGAAGAGAAAGGTGCTGTCGACGGCCCCC-’3 and VP64 reverse: 5′CTCGAGTCAGTTAATCACATGTCCAGG-′3.

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: .. The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. LYN mutants were generated by site-directed mutagenesis.

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: .. The full-length ATG5 gene was amplified from genomic DNA of the HK-2 cell line by PCR and subcloned into the pcDNA3.1 vector (Invitrogen,V79020). .. The ATG5-K130R mutant replacing Lys130 with Arg was generated by PCR and cloned into the Xhol and Xbal sites of pcDNA3.1-HA.

    Article Title: Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus
    Article Snippet: .. The non-structural NS3 gene of the dengue virus local isolate was cloned into pcDNA 3.1 Mammalian Expression Vector (Invitrogen, USA) by ligation of the amplified NS3 gene product into the digested vector and transformed into the bacterial cells. .. The NS3 mammalian expression vector constructs are shown in .

    Stable Transfection:

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. A stable cell line expressing each luciferase gene was established in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen) containing 10% foetal bovine serum (FBS) and 1 mg mL–1 geneticin for selection of stably transfected cells.

    Article Title: HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924
    Article Snippet: Paragraph title: Construction of a stable cell line ... The pcDNA 3.1 mammalian expression vector was purchased from Invitrogen (Carlsbad, CA). pcDNA 3.1 vector containing wild-type EGFR was a gift from C. David James. pcDNA 3.1 vector containing wild-type HER2 was a gift from Tai Wong.

    Article Title: Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus
    Article Snippet: Paragraph title: Development of stable cell line expressing dengue NS3 gene. Construction of NS3 mammalian expression vector ... The non-structural NS3 gene of the dengue virus local isolate was cloned into pcDNA 3.1 Mammalian Expression Vector (Invitrogen, USA) by ligation of the amplified NS3 gene product into the digested vector and transformed into the bacterial cells.

    Article Title: Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation
    Article Snippet: Sox2 ectopic expression was achieved by subcloning Sox2 cDNA from pCMV6-XL5- (OriGene) into pcDNA 3.1 mammalian expression vector (Invitrogen), under control of constitutive CMV promoter; the empty vector was used as control. .. Plasmid DNA constructs were verified by DNA sequencing and stably transfected into GBM monolayer cells using Lipofectamine 2000 (Invitrogen).

    Synthesized:

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies
    Article Snippet: .. The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR). ..

    Article Title: Krüppel-like factor 4 ameliorates diabetic kidney disease by activating autophagy via the mTOR pathway
    Article Snippet: .. KLF4 vector construction and transfection The synthetic gene fragments of KLF4 (synthesized by Shanghai Jierui Biological Engineering Co., Ltd.) were incorporated into a plasmid (pcDNA3.1; cat. no. V79020; Invitrogen; Thermo Fisher Scientific, Inc.) following double enzyme digestion with Nhe I-Bam HI (cat. nos. .. D1162A and D1010A; Takara Biotechnology Co., Ltd.), and termed pcDNA3.1(+)-KLF4.

    Construct:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: Paragraph title: Plasmid constructs and transfection ... The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020).

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The luciferase expression vector construct was transfected into U2OS human osteosarcoma cells (ATCC, Manassas, VA, USA) using the FuGene HD transfection reagent (Roche, Basel, Switzerland).

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies
    Article Snippet: .. The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR). ..

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators
    Article Snippet: Briefly, the corresponding TALE DNA-binding domain was constructed and cloned into pTAL destination vector using Golden Gate cloning method. .. The TALE DNA-binding domain was excised from pTAL using EcoRI and Bam H1 and subsequently subcloned into pcDNA-3.1 mammalian expression vector containing a CMV promoter (Invitrogen).

    Article Title: hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33
    Article Snippet: Paragraph title: Cell culture and h HSS1 expression vector construct ... The h HSS1 cDNA was subcloned from pTT3 vector into the Eco RI and Hin dIII sites of pcDNA.3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA).

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. U2OS cells were cultured and transfected in 6-well plates with 1 μg of LYN plasmids, which were cotransfected with 1 μg of a green fluorescent protein (GFP) construct (transfection efficiency control), with Turbofect (Fermentas).

    Article Title: Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus
    Article Snippet: The non-structural NS3 gene of the dengue virus local isolate was cloned into pcDNA 3.1 Mammalian Expression Vector (Invitrogen, USA) by ligation of the amplified NS3 gene product into the digested vector and transformed into the bacterial cells. .. The NS3 mammalian expression vector constructs are shown in .

    Luciferase:

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: .. Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The luciferase expression vector construct was transfected into U2OS human osteosarcoma cells (ATCC, Manassas, VA, USA) using the FuGene HD transfection reagent (Roche, Basel, Switzerland).

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. For luciferase assays, pCMV Sport6 plasmids encoding full length cDNAs of CLOCK, BMAL1, PER1, PER2, CRY1, CRY2, KPNB1 was obtained from the Mammalian Gene Collection (Thermo Fisher Scientific, Grand Island, NY, United States).

    Expressing:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: .. The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020). .. Scrambled and ATG5 -specific siRNA products were from Shanghai GenePharma Co., Ltd (Shanghai, China).

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: .. Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The luciferase expression vector construct was transfected into U2OS human osteosarcoma cells (ATCC, Manassas, VA, USA) using the FuGene HD transfection reagent (Roche, Basel, Switzerland).

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies
    Article Snippet: .. The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR). ..

    Article Title: Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors
    Article Snippet: .. The SNAP RNA isolation kit and pcDNA 3.1(−) mammalian expression vector were purchased from Invitrogen (Carlsbad, CA, U.S.A.). .. T4 DNA polymerase, T4 DNA ligase and Wizard® DNA clean-up system were purchased from Promega.

    Article Title: HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924
    Article Snippet: .. The pcDNA 3.1 mammalian expression vector was purchased from Invitrogen (Carlsbad, CA). pcDNA 3.1 vector containing wild-type EGFR was a gift from C. David James. pcDNA 3.1 vector containing wild-type HER2 was a gift from Tai Wong. .. The vectors were amplified transforming chemically competent E. coli and selecting on LB + ampicillin culture plates.

    Article Title: FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes
    Article Snippet: .. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures. .. Recombinant human FAM3B protein assays In order to verify the effects of exogenous FAM3B on cell viability 1 × 106 DU145 cells were seeded onto 2 cm diameter dishes and treated with recombinant human FAM3B protein (rhFAM3B) obtained in bacterial host.

    Article Title: Mechanism of RPE Cell Death in ?-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux
    Article Snippet: .. The PCR products were digested with EcoR1 and Zho1, and then ligated into pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, USA) having a neomycin resistance gene for selection. .. Sequences were confirmed by DNA sequencing in the core facility of the Norris Cancer Center of the University of Southern California.

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators
    Article Snippet: .. The TALE DNA-binding domain was excised from pTAL using EcoRI and Bam H1 and subsequently subcloned into pcDNA-3.1 mammalian expression vector containing a CMV promoter (Invitrogen). .. The native TALE activation domain was replaced with four copies of the minimal VP16 transactivation domain, VP64.

    Article Title: hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33
    Article Snippet: .. The h HSS1 cDNA was subcloned from pTT3 vector into the Eco RI and Hin dIII sites of pcDNA.3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The construct had a 6-His tag in-frame fused at the C-terminal of h HSS1 gene.

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: .. The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. LYN mutants were generated by site-directed mutagenesis.

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: .. Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. Plasmids encoding several flag-tagged clock proteins (Flag-PER1, Flag-PER2, Flag-CRY1, Flag-CRY2, Flag-CLOCK, Flag-REVERBα, Flag-CHRONO) were generated as described previously ( ).

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: The full-length ATG5 gene was amplified from genomic DNA of the HK-2 cell line by PCR and subcloned into the pcDNA3.1 vector (Invitrogen,V79020). .. The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020).

    Article Title: Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus
    Article Snippet: .. The non-structural NS3 gene of the dengue virus local isolate was cloned into pcDNA 3.1 Mammalian Expression Vector (Invitrogen, USA) by ligation of the amplified NS3 gene product into the digested vector and transformed into the bacterial cells. .. The NS3 mammalian expression vector constructs are shown in .

    Modification:

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. A stable cell line expressing each luciferase gene was established in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen) containing 10% foetal bovine serum (FBS) and 1 mg mL–1 geneticin for selection of stably transfected cells.

    Article Title: Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors
    Article Snippet: Polymerase chain reaction (PCR) primers, low and high glucose Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), Moloney murine leukaemia virus (MMLV) reverse transcriptase, oligo (dT)12 – 18 , random primers DNA labelling kits, LIPOFECTIN® reagent, neomycin (G418), Eco RV, Not I and Sac II were purchased from Life Technologies (Burlington, Ontario, Canada). .. The SNAP RNA isolation kit and pcDNA 3.1(−) mammalian expression vector were purchased from Invitrogen (Carlsbad, CA, U.S.A.).

    Transformation Assay:

    Article Title: Krüppel-like factor 4 ameliorates diabetic kidney disease by activating autophagy via the mTOR pathway
    Article Snippet: KLF4 vector construction and transfection The synthetic gene fragments of KLF4 (synthesized by Shanghai Jierui Biological Engineering Co., Ltd.) were incorporated into a plasmid (pcDNA3.1; cat. no. V79020; Invitrogen; Thermo Fisher Scientific, Inc.) following double enzyme digestion with Nhe I-Bam HI (cat. nos. .. The products were transformed into DH5α-competent cells (cat. no. CD201-01; Beijing TransGen Biotech Co., Ltd.), smeared on a lysogeny broth (LB) ampicillin (AMP) plate, and cultured at 37°C overnight.

    Article Title: Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus
    Article Snippet: .. The non-structural NS3 gene of the dengue virus local isolate was cloned into pcDNA 3.1 Mammalian Expression Vector (Invitrogen, USA) by ligation of the amplified NS3 gene product into the digested vector and transformed into the bacterial cells. .. The NS3 mammalian expression vector constructs are shown in .

    Over Expression:

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: .. Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. Plasmids encoding several flag-tagged clock proteins (Flag-PER1, Flag-PER2, Flag-CRY1, Flag-CRY2, Flag-CLOCK, Flag-REVERBα, Flag-CHRONO) were generated as described previously ( ).

    Transfection:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: Paragraph title: Plasmid constructs and transfection ... The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020).

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The luciferase expression vector construct was transfected into U2OS human osteosarcoma cells (ATCC, Manassas, VA, USA) using the FuGene HD transfection reagent (Roche, Basel, Switzerland).

    Article Title: HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924
    Article Snippet: The pcDNA 3.1 mammalian expression vector was purchased from Invitrogen (Carlsbad, CA). pcDNA 3.1 vector containing wild-type EGFR was a gift from C. David James. pcDNA 3.1 vector containing wild-type HER2 was a gift from Tai Wong. .. Midi scale DNA preparations were then made (Qiagen) and used for mammalian cell transfection of MCF-7 cells using Optifect Reagent, per the product instructions.

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. U2OS cells were cultured and transfected in 6-well plates with 1 μg of LYN plasmids, which were cotransfected with 1 μg of a green fluorescent protein (GFP) construct (transfection efficiency control), with Turbofect (Fermentas).

    Article Title: Krüppel-like factor 4 ameliorates diabetic kidney disease by activating autophagy via the mTOR pathway
    Article Snippet: .. KLF4 vector construction and transfection The synthetic gene fragments of KLF4 (synthesized by Shanghai Jierui Biological Engineering Co., Ltd.) were incorporated into a plasmid (pcDNA3.1; cat. no. V79020; Invitrogen; Thermo Fisher Scientific, Inc.) following double enzyme digestion with Nhe I-Bam HI (cat. nos. .. D1162A and D1010A; Takara Biotechnology Co., Ltd.), and termed pcDNA3.1(+)-KLF4.

    Article Title: Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation
    Article Snippet: Sox2 knockdown in low-passage 10% FBS cells was achieved by oligonucleotides targeting human SOX2-coding or nonsilencing control sequences cloned into BLOCK-iT Pol II miR RNAi expression vectors (Life Technologies) before cell transfection with Lipofectamine 2000 (Life Technologies). .. Sox2 ectopic expression was achieved by subcloning Sox2 cDNA from pCMV6-XL5- (OriGene) into pcDNA 3.1 mammalian expression vector (Invitrogen), under control of constitutive CMV promoter; the empty vector was used as control.

    Ligation:

    Article Title: Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus
    Article Snippet: .. The non-structural NS3 gene of the dengue virus local isolate was cloned into pcDNA 3.1 Mammalian Expression Vector (Invitrogen, USA) by ligation of the amplified NS3 gene product into the digested vector and transformed into the bacterial cells. .. The NS3 mammalian expression vector constructs are shown in .

    Cell Culture:

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. U2OS cells stably expressing the luciferase gene were cultured on 35 mm glass-bottomed dishes in DMEM containing 10% FBS.

    Article Title: hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33
    Article Snippet: Paragraph title: Cell culture and h HSS1 expression vector construct ... The h HSS1 cDNA was subcloned from pTT3 vector into the Eco RI and Hin dIII sites of pcDNA.3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA).

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. U2OS cells were cultured and transfected in 6-well plates with 1 μg of LYN plasmids, which were cotransfected with 1 μg of a green fluorescent protein (GFP) construct (transfection efficiency control), with Turbofect (Fermentas).

    Article Title: Krüppel-like factor 4 ameliorates diabetic kidney disease by activating autophagy via the mTOR pathway
    Article Snippet: KLF4 vector construction and transfection The synthetic gene fragments of KLF4 (synthesized by Shanghai Jierui Biological Engineering Co., Ltd.) were incorporated into a plasmid (pcDNA3.1; cat. no. V79020; Invitrogen; Thermo Fisher Scientific, Inc.) following double enzyme digestion with Nhe I-Bam HI (cat. nos. .. The products were transformed into DH5α-competent cells (cat. no. CD201-01; Beijing TransGen Biotech Co., Ltd.), smeared on a lysogeny broth (LB) ampicillin (AMP) plate, and cultured at 37°C overnight.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes
    Article Snippet: Cloning of FAM3B cDNA into lentiviral and plasmidial vectors Human FAM3B was amplified by RT-PCR from human pancreatic islets mRNA. .. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures.

    Generated:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: The ATG5-K130R mutant replacing Lys130 with Arg was generated by PCR and cloned into the Xhol and Xbal sites of pcDNA3.1-HA. .. The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020).

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. LYN mutants were generated by site-directed mutagenesis.

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. Plasmids encoding several flag-tagged clock proteins (Flag-PER1, Flag-PER2, Flag-CRY1, Flag-CRY2, Flag-CLOCK, Flag-REVERBα, Flag-CHRONO) were generated as described previously ( ).

    DNA Sequencing:

    Article Title: HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924
    Article Snippet: The pcDNA 3.1 mammalian expression vector was purchased from Invitrogen (Carlsbad, CA). pcDNA 3.1 vector containing wild-type EGFR was a gift from C. David James. pcDNA 3.1 vector containing wild-type HER2 was a gift from Tai Wong. .. The appropriate vector clones were then verified by diagnostic (Hind III/Xba I) digesting small scale mini preparations (Promega) and then verified by DNA sequencing.

    Article Title: Mechanism of RPE Cell Death in ?-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux
    Article Snippet: The PCR products were digested with EcoR1 and Zho1, and then ligated into pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, USA) having a neomycin resistance gene for selection. .. Sequences were confirmed by DNA sequencing in the core facility of the Norris Cancer Center of the University of Southern California.

    Article Title: Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation
    Article Snippet: Sox2 ectopic expression was achieved by subcloning Sox2 cDNA from pCMV6-XL5- (OriGene) into pcDNA 3.1 mammalian expression vector (Invitrogen), under control of constitutive CMV promoter; the empty vector was used as control. .. Plasmid DNA constructs were verified by DNA sequencing and stably transfected into GBM monolayer cells using Lipofectamine 2000 (Invitrogen).

    Sequencing:

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies
    Article Snippet: Molecular construct We used a MasR expression construct that consisted of the cDNA sequence corresponding to the human MasR (accession number: NM_002377.2) fused to the c-myc tag peptide at N-terminal. .. The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR).

    Article Title: FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes
    Article Snippet: Clones having appropriately sized inserts after digestion with restriction enzymes were sequenced using the T7 promoter primer (ABI PRISM Big Dye Terminator cycle sequencing kit, Applied Biosystems, Branchburg, NJ). .. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures.

    Article Title: hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33
    Article Snippet: The h HSS1 cDNA was subcloned from pTT3 vector into the Eco RI and Hin dIII sites of pcDNA.3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The resultant construct pCDNA3.1-h HSS1 was verified by sequencing analysis.

    Imaging:

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: .. Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The luciferase expression vector construct was transfected into U2OS human osteosarcoma cells (ATCC, Manassas, VA, USA) using the FuGene HD transfection reagent (Roche, Basel, Switzerland).

    Nucleic Acid Electrophoresis:

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. The cells were lysed 48 hours after transfection and cell lysates were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

    Mutagenesis:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: .. The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020). .. Scrambled and ATG5 -specific siRNA products were from Shanghai GenePharma Co., Ltd (Shanghai, China).

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. LYN mutants were generated by site-directed mutagenesis.

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: The full-length ATG5 gene was amplified from genomic DNA of the HK-2 cell line by PCR and subcloned into the pcDNA3.1 vector (Invitrogen,V79020). .. The ATG5-K130R mutant replacing Lys130 with Arg was generated by PCR and cloned into the Xhol and Xbal sites of pcDNA3.1-HA.

    Isolation:

    Article Title: Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors
    Article Snippet: .. The SNAP RNA isolation kit and pcDNA 3.1(−) mammalian expression vector were purchased from Invitrogen (Carlsbad, CA, U.S.A.). .. T4 DNA polymerase, T4 DNA ligase and Wizard® DNA clean-up system were purchased from Promega.

    Subcloning:

    Article Title: Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation
    Article Snippet: .. Sox2 ectopic expression was achieved by subcloning Sox2 cDNA from pCMV6-XL5- (OriGene) into pcDNA 3.1 mammalian expression vector (Invitrogen), under control of constitutive CMV promoter; the empty vector was used as control. .. Plasmid DNA constructs were verified by DNA sequencing and stably transfected into GBM monolayer cells using Lipofectamine 2000 (Invitrogen).

    Bimolecular Fluorescence Complementation Assay:

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. For plasmids expressing Venus-tagged PER1, PER2, CRY1, CRY2 (PER1-Venus, PER2-Venus, CRY1-Venus, CRY2-Venus) or BiFC plasmids expressing Venus N terminal (VN) or C-terminal (VC)-tagged PER1, PER2, CRY1, and CRY2 (PER1-VN, PER2-VN, CRY1-VC, CRY2-VC), full-length DNA fragment of the each of genes was subcloned into pCMV-Venus, pCMV-VN, or pCMV-VC using a restriction-free cloning method as described previously ( ; ).

    Purification:

    Article Title: FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes
    Article Snippet: PCR products were purified from agarose gels after electrophoretic separation and cloned into pGEM-T vector (Promega, Madison, WI). .. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures.

    Polymerase Chain Reaction:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: .. The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020). .. Scrambled and ATG5 -specific siRNA products were from Shanghai GenePharma Co., Ltd (Shanghai, China).

    Article Title: Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors
    Article Snippet: Polymerase chain reaction (PCR) primers, low and high glucose Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), Moloney murine leukaemia virus (MMLV) reverse transcriptase, oligo (dT)12 – 18 , random primers DNA labelling kits, LIPOFECTIN® reagent, neomycin (G418), Eco RV, Not I and Sac II were purchased from Life Technologies (Burlington, Ontario, Canada). .. The SNAP RNA isolation kit and pcDNA 3.1(−) mammalian expression vector were purchased from Invitrogen (Carlsbad, CA, U.S.A.).

    Article Title: FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes
    Article Snippet: PCR products were purified from agarose gels after electrophoretic separation and cloned into pGEM-T vector (Promega, Madison, WI). .. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures.

    Article Title: Mechanism of RPE Cell Death in ?-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux
    Article Snippet: .. The PCR products were digested with EcoR1 and Zho1, and then ligated into pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, USA) having a neomycin resistance gene for selection. .. Sequences were confirmed by DNA sequencing in the core facility of the Norris Cancer Center of the University of Southern California.

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators
    Article Snippet: The TALE DNA-binding domain was excised from pTAL using EcoRI and Bam H1 and subsequently subcloned into pcDNA-3.1 mammalian expression vector containing a CMV promoter (Invitrogen). .. The VP64 DNA fragment was PCR amplified from plasmid pLenti-EF1a-Backbone (NI) obtained from addgene ( http://www.addgene.org/27962 ) and a SV40 nuclear localization signal added to N-terminus of VP64 using primers NLS_VP64 forward: 5′CCGCGGAGCCCCAAGAAGAAGAGAAAGGTGCTGTCGACGGCCCCC-’3 and VP64 reverse: 5′CTCGAGTCAGTTAATCACATGTCCAGG-′3.

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: .. Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. Plasmids encoding several flag-tagged clock proteins (Flag-PER1, Flag-PER2, Flag-CRY1, Flag-CRY2, Flag-CLOCK, Flag-REVERBα, Flag-CHRONO) were generated as described previously ( ).

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: .. The full-length ATG5 gene was amplified from genomic DNA of the HK-2 cell line by PCR and subcloned into the pcDNA3.1 vector (Invitrogen,V79020). .. The ATG5-K130R mutant replacing Lys130 with Arg was generated by PCR and cloned into the Xhol and Xbal sites of pcDNA3.1-HA.

    Blocking Assay:

    Article Title: Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation
    Article Snippet: Sox2 knockdown in low-passage 10% FBS cells was achieved by oligonucleotides targeting human SOX2-coding or nonsilencing control sequences cloned into BLOCK-iT Pol II miR RNAi expression vectors (Life Technologies) before cell transfection with Lipofectamine 2000 (Life Technologies). .. Sox2 ectopic expression was achieved by subcloning Sox2 cDNA from pCMV6-XL5- (OriGene) into pcDNA 3.1 mammalian expression vector (Invitrogen), under control of constitutive CMV promoter; the empty vector was used as control.

    SDS Page:

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. The cells were lysed 48 hours after transfection and cell lysates were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

    Plasmid Preparation:

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: .. The primers used for PCR were ATG5 : sense 5′-GCGTCGACTCCTGGAAGAATGACAGAT-3′, antisense 5′-CGCCTCGAGTCCTTCAATCTGTTGGCT-3′ and ATG5-K130R mutant: sense 5′-CATGTATGAGAGAAGCTGATGCT-3′, antisense 3′-AGCATCAGCTTCTCTCATACATGA-5′. pcDNA3.1-HA mammalian expression vector was from Invitrogen Life Technologies (V79020). .. Scrambled and ATG5 -specific siRNA products were from Shanghai GenePharma Co., Ltd (Shanghai, China).

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: .. Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The luciferase expression vector construct was transfected into U2OS human osteosarcoma cells (ATCC, Manassas, VA, USA) using the FuGene HD transfection reagent (Roche, Basel, Switzerland).

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies
    Article Snippet: .. The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR). ..

    Article Title: Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors
    Article Snippet: .. The SNAP RNA isolation kit and pcDNA 3.1(−) mammalian expression vector were purchased from Invitrogen (Carlsbad, CA, U.S.A.). .. T4 DNA polymerase, T4 DNA ligase and Wizard® DNA clean-up system were purchased from Promega.

    Article Title: HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924
    Article Snippet: .. The pcDNA 3.1 mammalian expression vector was purchased from Invitrogen (Carlsbad, CA). pcDNA 3.1 vector containing wild-type EGFR was a gift from C. David James. pcDNA 3.1 vector containing wild-type HER2 was a gift from Tai Wong. .. The vectors were amplified transforming chemically competent E. coli and selecting on LB + ampicillin culture plates.

    Article Title: FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes
    Article Snippet: .. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures. .. Recombinant human FAM3B protein assays In order to verify the effects of exogenous FAM3B on cell viability 1 × 106 DU145 cells were seeded onto 2 cm diameter dishes and treated with recombinant human FAM3B protein (rhFAM3B) obtained in bacterial host.

    Article Title: Mechanism of RPE Cell Death in ?-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux
    Article Snippet: .. The PCR products were digested with EcoR1 and Zho1, and then ligated into pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, USA) having a neomycin resistance gene for selection. .. Sequences were confirmed by DNA sequencing in the core facility of the Norris Cancer Center of the University of Southern California.

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators
    Article Snippet: .. The TALE DNA-binding domain was excised from pTAL using EcoRI and Bam H1 and subsequently subcloned into pcDNA-3.1 mammalian expression vector containing a CMV promoter (Invitrogen). .. The native TALE activation domain was replaced with four copies of the minimal VP16 transactivation domain, VP64.

    Article Title: hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33
    Article Snippet: .. The h HSS1 cDNA was subcloned from pTT3 vector into the Eco RI and Hin dIII sites of pcDNA.3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. The construct had a 6-His tag in-frame fused at the C-terminal of h HSS1 gene.

    Article Title: Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
    Article Snippet: .. The coding region of LYN was amplified from human cDNA and ligated into a pcDNA 3.1 mammalian expression vector (Life Technologies). .. LYN mutants were generated by site-directed mutagenesis.

    Article Title: Krüppel-like factor 4 ameliorates diabetic kidney disease by activating autophagy via the mTOR pathway
    Article Snippet: .. KLF4 vector construction and transfection The synthetic gene fragments of KLF4 (synthesized by Shanghai Jierui Biological Engineering Co., Ltd.) were incorporated into a plasmid (pcDNA3.1; cat. no. V79020; Invitrogen; Thermo Fisher Scientific, Inc.) following double enzyme digestion with Nhe I-Bam HI (cat. nos. .. D1162A and D1010A; Takara Biotechnology Co., Ltd.), and termed pcDNA3.1(+)-KLF4.

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: .. Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. Plasmids encoding several flag-tagged clock proteins (Flag-PER1, Flag-PER2, Flag-CRY1, Flag-CRY2, Flag-CLOCK, Flag-REVERBα, Flag-CHRONO) were generated as described previously ( ).

    Article Title: Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis
    Article Snippet: .. The full-length ATG5 gene was amplified from genomic DNA of the HK-2 cell line by PCR and subcloned into the pcDNA3.1 vector (Invitrogen,V79020). .. The ATG5-K130R mutant replacing Lys130 with Arg was generated by PCR and cloned into the Xhol and Xbal sites of pcDNA3.1-HA.

    Article Title: Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus
    Article Snippet: .. The non-structural NS3 gene of the dengue virus local isolate was cloned into pcDNA 3.1 Mammalian Expression Vector (Invitrogen, USA) by ligation of the amplified NS3 gene product into the digested vector and transformed into the bacterial cells. .. The NS3 mammalian expression vector constructs are shown in .

    Selection:

    Article Title: Bioluminescence microscopy using a short focal-length imaging lens
    Article Snippet: Bioluminescence imaging Luciferase genes originating from the firefly (Luc2), click beetle (CBG99 and CBR) or deep-sea shrimp (NanoLuc; Promega, Madison, WI, USA) were inserted into the pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). .. A stable cell line expressing each luciferase gene was established in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen) containing 10% foetal bovine serum (FBS) and 1 mg mL–1 geneticin for selection of stably transfected cells.

    Article Title: Mechanism of RPE Cell Death in ?-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux
    Article Snippet: .. The PCR products were digested with EcoR1 and Zho1, and then ligated into pcDNA 3.1 mammalian expression vector (Invitrogen, Carlsbad, USA) having a neomycin resistance gene for selection. .. Sequences were confirmed by DNA sequencing in the core facility of the Norris Cancer Center of the University of Southern California.

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators
    Article Snippet: Paragraph title: Construction of TALEs and Target Site Selection ... The TALE DNA-binding domain was excised from pTAL using EcoRI and Bam H1 and subsequently subcloned into pcDNA-3.1 mammalian expression vector containing a CMV promoter (Invitrogen).

    Transgenic Assay:

    Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function
    Article Snippet: .. Plasmids For overexpression of NRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). .. Plasmids encoding several flag-tagged clock proteins (Flag-PER1, Flag-PER2, Flag-CRY1, Flag-CRY2, Flag-CLOCK, Flag-REVERBα, Flag-CHRONO) were generated as described previously ( ).

    Activation Assay:

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators
    Article Snippet: The TALE DNA-binding domain was excised from pTAL using EcoRI and Bam H1 and subsequently subcloned into pcDNA-3.1 mammalian expression vector containing a CMV promoter (Invitrogen). .. The native TALE activation domain was replaced with four copies of the minimal VP16 transactivation domain, VP64.

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  • 90
    Thermo Fisher pcdna3 1
    PR130 expression antagonises ATM phosphorylation. a Schematic of PPP2R3A knockout using CRISPR-Cas9 technology. Humanised S. pyogenes Cas9 nuclease was expressed in HCT116 cells with two guide RNAs (gRNA) to introduce two DSB into the PPP2R3A gene (684 bp apart) as described in the Methods section. The usage of two cleavage sites increased the chances to create a successful knockout. b HCT116 ΔgRNA and HCT116 ΔPR130 (clone #16) cells were cultured with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. ATM, pATM (S1981), p53, pp53 (S15) and PR130 were detected by western blot. β-Actin served as loading control. Numbers indicate densitometric analysis of pATM signal relative to HU-treated sample of each cell line and normalised to β-Actin signal ( n = 3). c HCT116 cells were transiently transfected with indicated siRNAs. After 24 h, cells were treated with 1 mM HU and 2 µM MS-275 (24 h). Protein detection was performed by immunoblot ( n = 2). d After 20 h of treatment with HU (1 mM) and MS-275 (2 µM), okadaic acid (OA, 25 nM) was added for further 4 h. Co-immunoprecipitations (IP) with anti-pS1981-ATM antibody, rabbit pre-immune serum (pre) or no antibody (no AB) were analysed for pATM and PR130 by western blot. Input (IN) represents 6% of the lysates used for IP. The right panel shows results that were obtained with the same strategy, including individual treatment with HU and MS-275. Data represent results from three independent experiments that were carried out in a similar manner. e IP performed with PR130 antibody using lysates from untreated and MS-275-treated HCT116. Precipitates were probed with pan-acetyl-lysine (ac-Lys) and PR130 antibody. Input (IN) is 6% of the lysate used for immunoprecipitation ( n = 2). Numbers indicate densitometric analysis of ac-Lys and PR130 signal relative to untreated cells. f HCT116 cells were transfected with HA-PR130 or vector control <t>(pcDNA3.1)</t> for 24 h and subsequently treated with MS-275 (2 µM, 24 h). Phosphatase activity of the HA-precipitates against phospho-S1981-ATM peptide or a threonine phosphopeptide (positive control) were measured in vitro as liberated pmol phosphate/min. Data are presented as mean ± SEM ( n = 3). The western blot verifies precipitation of HA-tagged PR130
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1/product/Thermo Fisher
    Average 90 stars, based on 503 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    90
    Thermo Fisher pcdna3 1 vector
    Fgfr AS is regulated by Esrp genes in vertebrates and amphioxus. a RT-PCR assays showing differential Fgfr exon IIIb and IIIc inclusion in WT versus DMUT 5 d.p.f. zebrafish embryos. b RT-PCR assays for Fgfr AS in different amphioxus adult tissues, depicted in a transversal section. nc, nerve cord, ms, muscle, gl, gills, hd, hepatic diverticulum, nt, notochord, sk, skin. Reverse primers were designed in both exons IIIb and IIIc (arrows) and used together in the same PCR reaction. c Top: schematic representation of <t>pcDNA3.1-based</t> minigene constructs containing the genomic region spanning the Fgfr AS event of Branchiostoma lanceolatum , with (pcDNA3.1-BlaFGFR) and without (pcDNA3.1-BlaFGFRΔIIIx) exon IIIx. Bottom: relative intensity of fluorescent RT-PCR bands supporting differential inclusion of exons IIIb and IIIc when transfecting the minigenes alone (Control) or together with a plasmid containing either amphioxus or zebrafish full-length Esrp transcripts (BlaEsrp and DreEsrp1, respectively). Despite significant mis-splicing of the minigene in all conditions, only the amphioxus construct was able to induce a dramatic switch toward exon IIIb inclusion. Primers were designed in the neighboring constitutive exons (arrows). d RT-PCR assays for endogenous human AS events in the same control, BlaEsrp or DreEsrp1 transfected 293T cells showing that the amphioxus and zebrafish Esrp constructs are able to modulate endogenous Esrp -dependent events in a similar manner. Error bars correspond to standard errors of three biological replicates. Esrp -enhanced isoforms are marked with an isoform cartoon
    Pcdna3 1 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 605 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 vector/product/Thermo Fisher
    Average 90 stars, based on 605 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 vector - by Bioz Stars, 2020-02
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    This pcDNA 3 1 Zeo vector is designed for high level constitutive expression in a variety of mammalian cell lines It contains a Zeocin selectable marker and a reverse orientation
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    PR130 expression antagonises ATM phosphorylation. a Schematic of PPP2R3A knockout using CRISPR-Cas9 technology. Humanised S. pyogenes Cas9 nuclease was expressed in HCT116 cells with two guide RNAs (gRNA) to introduce two DSB into the PPP2R3A gene (684 bp apart) as described in the Methods section. The usage of two cleavage sites increased the chances to create a successful knockout. b HCT116 ΔgRNA and HCT116 ΔPR130 (clone #16) cells were cultured with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. ATM, pATM (S1981), p53, pp53 (S15) and PR130 were detected by western blot. β-Actin served as loading control. Numbers indicate densitometric analysis of pATM signal relative to HU-treated sample of each cell line and normalised to β-Actin signal ( n = 3). c HCT116 cells were transiently transfected with indicated siRNAs. After 24 h, cells were treated with 1 mM HU and 2 µM MS-275 (24 h). Protein detection was performed by immunoblot ( n = 2). d After 20 h of treatment with HU (1 mM) and MS-275 (2 µM), okadaic acid (OA, 25 nM) was added for further 4 h. Co-immunoprecipitations (IP) with anti-pS1981-ATM antibody, rabbit pre-immune serum (pre) or no antibody (no AB) were analysed for pATM and PR130 by western blot. Input (IN) represents 6% of the lysates used for IP. The right panel shows results that were obtained with the same strategy, including individual treatment with HU and MS-275. Data represent results from three independent experiments that were carried out in a similar manner. e IP performed with PR130 antibody using lysates from untreated and MS-275-treated HCT116. Precipitates were probed with pan-acetyl-lysine (ac-Lys) and PR130 antibody. Input (IN) is 6% of the lysate used for immunoprecipitation ( n = 2). Numbers indicate densitometric analysis of ac-Lys and PR130 signal relative to untreated cells. f HCT116 cells were transfected with HA-PR130 or vector control (pcDNA3.1) for 24 h and subsequently treated with MS-275 (2 µM, 24 h). Phosphatase activity of the HA-precipitates against phospho-S1981-ATM peptide or a threonine phosphopeptide (positive control) were measured in vitro as liberated pmol phosphate/min. Data are presented as mean ± SEM ( n = 3). The western blot verifies precipitation of HA-tagged PR130

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: PR130 expression antagonises ATM phosphorylation. a Schematic of PPP2R3A knockout using CRISPR-Cas9 technology. Humanised S. pyogenes Cas9 nuclease was expressed in HCT116 cells with two guide RNAs (gRNA) to introduce two DSB into the PPP2R3A gene (684 bp apart) as described in the Methods section. The usage of two cleavage sites increased the chances to create a successful knockout. b HCT116 ΔgRNA and HCT116 ΔPR130 (clone #16) cells were cultured with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. ATM, pATM (S1981), p53, pp53 (S15) and PR130 were detected by western blot. β-Actin served as loading control. Numbers indicate densitometric analysis of pATM signal relative to HU-treated sample of each cell line and normalised to β-Actin signal ( n = 3). c HCT116 cells were transiently transfected with indicated siRNAs. After 24 h, cells were treated with 1 mM HU and 2 µM MS-275 (24 h). Protein detection was performed by immunoblot ( n = 2). d After 20 h of treatment with HU (1 mM) and MS-275 (2 µM), okadaic acid (OA, 25 nM) was added for further 4 h. Co-immunoprecipitations (IP) with anti-pS1981-ATM antibody, rabbit pre-immune serum (pre) or no antibody (no AB) were analysed for pATM and PR130 by western blot. Input (IN) represents 6% of the lysates used for IP. The right panel shows results that were obtained with the same strategy, including individual treatment with HU and MS-275. Data represent results from three independent experiments that were carried out in a similar manner. e IP performed with PR130 antibody using lysates from untreated and MS-275-treated HCT116. Precipitates were probed with pan-acetyl-lysine (ac-Lys) and PR130 antibody. Input (IN) is 6% of the lysate used for immunoprecipitation ( n = 2). Numbers indicate densitometric analysis of ac-Lys and PR130 signal relative to untreated cells. f HCT116 cells were transfected with HA-PR130 or vector control (pcDNA3.1) for 24 h and subsequently treated with MS-275 (2 µM, 24 h). Phosphatase activity of the HA-precipitates against phospho-S1981-ATM peptide or a threonine phosphopeptide (positive control) were measured in vitro as liberated pmol phosphate/min. Data are presented as mean ± SEM ( n = 3). The western blot verifies precipitation of HA-tagged PR130

    Article Snippet: HCT116 cells were transfected with HA-tagged PR130 or pcDNA3.1 (vector control) using TurboFect (ThermoFisher Scientific).

    Techniques: Expressing, Knock-Out, CRISPR, Introduce, Cell Culture, Mass Spectrometry, Western Blot, Transfection, Immunoprecipitation, Plasmid Preparation, Activity Assay, Positive Control, In Vitro

    SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection, Immunoprecipitation, SDS Page

    SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Purification, Transfection, Produced

    SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Expressing, Transfection, Plasmid Preparation, Standard Deviation, Activity Assay, Luciferase, Protein Concentration

    Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection

    SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection, Standard Deviation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

    Fgfr AS is regulated by Esrp genes in vertebrates and amphioxus. a RT-PCR assays showing differential Fgfr exon IIIb and IIIc inclusion in WT versus DMUT 5 d.p.f. zebrafish embryos. b RT-PCR assays for Fgfr AS in different amphioxus adult tissues, depicted in a transversal section. nc, nerve cord, ms, muscle, gl, gills, hd, hepatic diverticulum, nt, notochord, sk, skin. Reverse primers were designed in both exons IIIb and IIIc (arrows) and used together in the same PCR reaction. c Top: schematic representation of pcDNA3.1-based minigene constructs containing the genomic region spanning the Fgfr AS event of Branchiostoma lanceolatum , with (pcDNA3.1-BlaFGFR) and without (pcDNA3.1-BlaFGFRΔIIIx) exon IIIx. Bottom: relative intensity of fluorescent RT-PCR bands supporting differential inclusion of exons IIIb and IIIc when transfecting the minigenes alone (Control) or together with a plasmid containing either amphioxus or zebrafish full-length Esrp transcripts (BlaEsrp and DreEsrp1, respectively). Despite significant mis-splicing of the minigene in all conditions, only the amphioxus construct was able to induce a dramatic switch toward exon IIIb inclusion. Primers were designed in the neighboring constitutive exons (arrows). d RT-PCR assays for endogenous human AS events in the same control, BlaEsrp or DreEsrp1 transfected 293T cells showing that the amphioxus and zebrafish Esrp constructs are able to modulate endogenous Esrp -dependent events in a similar manner. Error bars correspond to standard errors of three biological replicates. Esrp -enhanced isoforms are marked with an isoform cartoon

    Journal: Nature Communications

    Article Title: Evolutionary recruitment of flexible Esrp-dependent splicing programs into diverse embryonic morphogenetic processes

    doi: 10.1038/s41467-017-01961-y

    Figure Lengend Snippet: Fgfr AS is regulated by Esrp genes in vertebrates and amphioxus. a RT-PCR assays showing differential Fgfr exon IIIb and IIIc inclusion in WT versus DMUT 5 d.p.f. zebrafish embryos. b RT-PCR assays for Fgfr AS in different amphioxus adult tissues, depicted in a transversal section. nc, nerve cord, ms, muscle, gl, gills, hd, hepatic diverticulum, nt, notochord, sk, skin. Reverse primers were designed in both exons IIIb and IIIc (arrows) and used together in the same PCR reaction. c Top: schematic representation of pcDNA3.1-based minigene constructs containing the genomic region spanning the Fgfr AS event of Branchiostoma lanceolatum , with (pcDNA3.1-BlaFGFR) and without (pcDNA3.1-BlaFGFRΔIIIx) exon IIIx. Bottom: relative intensity of fluorescent RT-PCR bands supporting differential inclusion of exons IIIb and IIIc when transfecting the minigenes alone (Control) or together with a plasmid containing either amphioxus or zebrafish full-length Esrp transcripts (BlaEsrp and DreEsrp1, respectively). Despite significant mis-splicing of the minigene in all conditions, only the amphioxus construct was able to induce a dramatic switch toward exon IIIb inclusion. Primers were designed in the neighboring constitutive exons (arrows). d RT-PCR assays for endogenous human AS events in the same control, BlaEsrp or DreEsrp1 transfected 293T cells showing that the amphioxus and zebrafish Esrp constructs are able to modulate endogenous Esrp -dependent events in a similar manner. Error bars correspond to standard errors of three biological replicates. Esrp -enhanced isoforms are marked with an isoform cartoon

    Article Snippet: Amphioxus Fgfr AS minigenes and cell cultures The full-length Esrp open reading frame (ORF) from B. lanceolatum and esrp1 ORF from D. rerio were amplified from cDNA using iProof High Fidelity Polymerase (Bio-Rad) and cloned into the pcDNA3.1 vector (Thermo Fisher Scientific).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Polymerase Chain Reaction, Construct, Plasmid Preparation, Transfection

    UCA1 acted as a ceRNA of miR129 to enhance target SOX4 gene expression in RCC cells. Notes: ( A ) 786O cells were transfected with miR129 mimic or miR-Con, and ACHN cells were transfected with miR129 inhibitor or anti-miR-Con, followed by the detection of SOX4 protein expression. ( B ) 786O cells were transfected with siCon or siUCA1-2, and ACHN cells were transfected with pcDNA3.1 vector or pcDNA-UCA1 plasmid, followed by the determination of SOX4 protein expression. ( C ) Effects of miR129 and UCA1 overexpression on luciferase activity of SOX4 reporter were evaluated in 786O cells. ( D ) The effect of miR129 downregulation and UCA1 silencing on luciferase activity of SOX4 reporter was monitored in ACHN cells. ( E ) SOX4 mRNA expression in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( F ) Spearman’s correlation analysis of SOX4 mRNA and UCA1 expressions in 40 cases of RCC tumor tissue. * P

    Journal: OncoTargets and therapy

    Article Title: UCA1 promotes cell proliferation and invasion and inhibits apoptosis through regulation of the miR129–SOX4 pathway in renal cell carcinoma

    doi: 10.2147/OTT.S160192

    Figure Lengend Snippet: UCA1 acted as a ceRNA of miR129 to enhance target SOX4 gene expression in RCC cells. Notes: ( A ) 786O cells were transfected with miR129 mimic or miR-Con, and ACHN cells were transfected with miR129 inhibitor or anti-miR-Con, followed by the detection of SOX4 protein expression. ( B ) 786O cells were transfected with siCon or siUCA1-2, and ACHN cells were transfected with pcDNA3.1 vector or pcDNA-UCA1 plasmid, followed by the determination of SOX4 protein expression. ( C ) Effects of miR129 and UCA1 overexpression on luciferase activity of SOX4 reporter were evaluated in 786O cells. ( D ) The effect of miR129 downregulation and UCA1 silencing on luciferase activity of SOX4 reporter was monitored in ACHN cells. ( E ) SOX4 mRNA expression in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( F ) Spearman’s correlation analysis of SOX4 mRNA and UCA1 expressions in 40 cases of RCC tumor tissue. * P

    Article Snippet: Full-length sequences of UCA1 were amplified by polymerase chain reaction (PCR) and inserted into pcDNA3.1 vector (Thermo Fisher Scientific) to gain the UCA1-overexpression plasmid.

    Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay

    UCA1 inhibited miR129 expression in RCC cells by direct interaction. Notes: ( A ) Exhibition of putative binding sites between UCA1 and miR129 and mutant (Mut) sites in UCA1-Mut reporter. ( B ) 786O cells were cotransfected with miR control (Con) or miR129 mimic and wild-type (WT) UCA1 or UCA1-Mut reporter, followed by the detection of luciferase activity at 48 hours posttransfection. ( C ) Luciferase activity was measured in ACHN cells cotransfected with anti-miR-Con or miR129 inhibitor and UCA1-WT or UCA1-Mut reporter at 48 hours after transfection. ( D ) RNA pull-down assays were used to further validate the interaction between UCA1 and miR129 in 786O and ACHN cells. ( E , F ) RNA-immunoprecipitation assays were employed using Ago2 or IgG antibody to explore the enrichment degrees of UCA1 and miR129 in Ago2 or IgG immunoprecipitation complexes of 786O and ACHN cells. The input group acted as a positive control. ( G , H ) miR129 expression was determined in 786O and ACHN cells transfected with siCon ( G ), siUCA1-2 ( G ), UCA1 overexpression plasmid ( H ) or pcDNA3.1 empty vector ( H ). ( I ) miR129 expression analysis in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( J ) Spearman’s correlation analysis of miR129 and UCA1 expression in 40 cases of RCC tumor tissue. * P

    Journal: OncoTargets and therapy

    Article Title: UCA1 promotes cell proliferation and invasion and inhibits apoptosis through regulation of the miR129–SOX4 pathway in renal cell carcinoma

    doi: 10.2147/OTT.S160192

    Figure Lengend Snippet: UCA1 inhibited miR129 expression in RCC cells by direct interaction. Notes: ( A ) Exhibition of putative binding sites between UCA1 and miR129 and mutant (Mut) sites in UCA1-Mut reporter. ( B ) 786O cells were cotransfected with miR control (Con) or miR129 mimic and wild-type (WT) UCA1 or UCA1-Mut reporter, followed by the detection of luciferase activity at 48 hours posttransfection. ( C ) Luciferase activity was measured in ACHN cells cotransfected with anti-miR-Con or miR129 inhibitor and UCA1-WT or UCA1-Mut reporter at 48 hours after transfection. ( D ) RNA pull-down assays were used to further validate the interaction between UCA1 and miR129 in 786O and ACHN cells. ( E , F ) RNA-immunoprecipitation assays were employed using Ago2 or IgG antibody to explore the enrichment degrees of UCA1 and miR129 in Ago2 or IgG immunoprecipitation complexes of 786O and ACHN cells. The input group acted as a positive control. ( G , H ) miR129 expression was determined in 786O and ACHN cells transfected with siCon ( G ), siUCA1-2 ( G ), UCA1 overexpression plasmid ( H ) or pcDNA3.1 empty vector ( H ). ( I ) miR129 expression analysis in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( J ) Spearman’s correlation analysis of miR129 and UCA1 expression in 40 cases of RCC tumor tissue. * P

    Article Snippet: Full-length sequences of UCA1 were amplified by polymerase chain reaction (PCR) and inserted into pcDNA3.1 vector (Thermo Fisher Scientific) to gain the UCA1-overexpression plasmid.

    Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Immunoprecipitation, Positive Control, Over Expression, Plasmid Preparation