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  • 99
    ATCC 293 cells hek 293t
    Cdh2 on 293TA cells provides favorable surface for neurite branching of cocultured cortical neurons. (A–D,A′–D′) Mouse cortical neurons were cultured with <t>293T</t> derivatives and stained with anti-synapsin I (red) and phalloidin (green). (A) Cortical neurites frequently branch on 293TA cells as shown in the high-power picture (bottom; region indicated by blue arrow in top panel). (B) Neurites generally grew unbranched near the edge of 293NC cells (C) Neurites branched on Cdh2-overexpressing 293NC (D) Neurites failed to branch on neural cell adhesion molecule (NCAM)-overexpressing 293NC cells. Bar, 25 μm for (A–D) , and 5 μm for high-power images (bottom). (E) Percent of neurons exhibiting branching at sites of apposition with <t>293</t> cells ( n = 20 neurons per condition). Co-expression of NLGN1 had no effect on branching in the presence or absence of Cdh2. (F,G) Quantification of calcium-dependent (F) and independent (G) cell aggregation assays (Mean ± SEM, n = 10). Significance of differences at 60 min and 120 min, p
    293 Cells Hek 293t, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 293t cells
    The Arg649Glu mutation activates ALIX for membrane association and virus budding. (A) Flotation analysis showing the degree of membrane association of ALIX and ALIX R649E . Lanes 1 to 3 show the crude fractionation of <t>293T</t> cell lysates (lane 1) expressing
    293t Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7714 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ATCC hek 293t cells
    Design of the 5’ UTR library of naturally occurring and synthetic 5’ UTRs. RNA-seq and Ribo-seq datasets of <t>HEK</t> <t>293T,</t> PC3, and human muscle cells, together with the GTEx database of human muscle tissue, were collected. Natural 5’UTRs with high TEs and low TEs in HEK 293T and RD cells, 5’ UTRs with various TEs in human muscle cells, and the 5’ UTRs with high mRNA counts in human muscle tissues were selected and added to the library. In addition, we designed synthetic 5’ UTRs by: i) collecting endogenous 5’ UTR sequences on the target cell type (HEK 293T, PC3 or human muscle cells) from public data; ii) extracting sequence features of the 5’ UTRs, including those nucleotides surrounding the AUG region; iii) training a Random Forest machine learning method for each cell type/tissue (HEK 293T, PC3 or human muscle cells), to learn a function that maps sequence features to mRNA expression levels and TEs; and iv) designing a set of 100 bp synthetic sequences that are predicted to maximize TEs and protein expression levels using genetic algorithms.
    Hek 293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 5105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek 293 cells
    Membrane localization of EGFP-tagged Ca V 1.2e1b and Ca V 1.2e1c channels when expressed with α 2 δ or with α 2 δ + β 1b subunits in <t>HEK</t> 293 cells
    Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 6302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Proteintech human hek 293 cells
    Sp1 binds to the human Klotho gene ( KL ) promoter. ( a ) <t>HEK-293</t> cells were co-transfected with Full KL/LUC reporter and pcDNA3 or pcDNA3-Sp1 plasmid. 24 h after transfection, cells were lysed and transcriptional activity was detected by dual-luciferase assays. ( b ) Cells were transfected with full length or truncated reporter, combined with pcDNA3 or pcDNA3-Sp1 plasmid. 24 h after transfection, HEK-293 cells were lysed and transcriptional activity was detected. ( c ) HEK-293 cells were transfected with KL5/LUC or mutated KL5/LUC reporter, and transcriptional activity was detected as before. As an internal control, Rinella (pRL-TK) was used to normalize the transfection efficiency. Mean ± SD of triplicate experiments performed independently. *** p
    Human Hek 293 Cells, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC cell culture hek 293 t cells
    PMTb-GFP initially colocalizes with CTxB-594, but then diverges and is trafficked to and translocated from acidic endosomes. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q  as described in Methods. Seven h post-transfection cells were treated with ( a ) NH 4 Cl or ( b ) bafilomycin A1 (BAF) at the indicated concentrations for 15 min before treatment with 100 ng/mL rPMT. After 15 h incubation, cells were harvested and assayed for SRE reporter gene activity, as described in the Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT-treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of three experiments ± S.D. with each experiment performed in triplicate, where *  p
    Cell Culture Hek 293 T Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC human embryonic kidney hek293 cells
    Examination of the internalization and β-arrestin-binding properties of N321K-V2R. <t>HEK293</t> cells were transiently transfected with the plasmids of the indicated BRET partners and after 24 hours, the cells were exposed to AVP or vehicle (veh). A
    Human Embryonic Kidney Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 652 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa lentix hek 293 t cells
    Examination of the internalization and β-arrestin-binding properties of N321K-V2R. <t>HEK293</t> cells were transiently transfected with the plasmids of the indicated BRET partners and after 24 hours, the cells were exposed to AVP or vehicle (veh). A
    Lentix Hek 293 T Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cdh2 on 293TA cells provides favorable surface for neurite branching of cocultured cortical neurons. (A–D,A′–D′) Mouse cortical neurons were cultured with 293T derivatives and stained with anti-synapsin I (red) and phalloidin (green). (A) Cortical neurites frequently branch on 293TA cells as shown in the high-power picture (bottom; region indicated by blue arrow in top panel). (B) Neurites generally grew unbranched near the edge of 293NC cells (C) Neurites branched on Cdh2-overexpressing 293NC (D) Neurites failed to branch on neural cell adhesion molecule (NCAM)-overexpressing 293NC cells. Bar, 25 μm for (A–D) , and 5 μm for high-power images (bottom). (E) Percent of neurons exhibiting branching at sites of apposition with 293 cells ( n = 20 neurons per condition). Co-expression of NLGN1 had no effect on branching in the presence or absence of Cdh2. (F,G) Quantification of calcium-dependent (F) and independent (G) cell aggregation assays (Mean ± SEM, n = 10). Significance of differences at 60 min and 120 min, p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Cadherins Interact With Synaptic Organizers to Promote Synaptic Differentiation

    doi: 10.3389/fnmol.2018.00142

    Figure Lengend Snippet: Cdh2 on 293TA cells provides favorable surface for neurite branching of cocultured cortical neurons. (A–D,A′–D′) Mouse cortical neurons were cultured with 293T derivatives and stained with anti-synapsin I (red) and phalloidin (green). (A) Cortical neurites frequently branch on 293TA cells as shown in the high-power picture (bottom; region indicated by blue arrow in top panel). (B) Neurites generally grew unbranched near the edge of 293NC cells (C) Neurites branched on Cdh2-overexpressing 293NC (D) Neurites failed to branch on neural cell adhesion molecule (NCAM)-overexpressing 293NC cells. Bar, 25 μm for (A–D) , and 5 μm for high-power images (bottom). (E) Percent of neurons exhibiting branching at sites of apposition with 293 cells ( n = 20 neurons per condition). Co-expression of NLGN1 had no effect on branching in the presence or absence of Cdh2. (F,G) Quantification of calcium-dependent (F) and independent (G) cell aggregation assays (Mean ± SEM, n = 10). Significance of differences at 60 min and 120 min, p

    Article Snippet: 293 Cells HEK-293T were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Cell Culture, Staining, Expressing

    The Arg649Glu mutation activates ALIX for membrane association and virus budding. (A) Flotation analysis showing the degree of membrane association of ALIX and ALIX R649E . Lanes 1 to 3 show the crude fractionation of 293T cell lysates (lane 1) expressing

    Journal: Journal of Virology

    Article Title: Activation of the Retroviral Budding Factor ALIX ▿Activation of the Retroviral Budding Factor ALIX ▿ †

    doi: 10.1128/JVI.02653-10

    Figure Lengend Snippet: The Arg649Glu mutation activates ALIX for membrane association and virus budding. (A) Flotation analysis showing the degree of membrane association of ALIX and ALIX R649E . Lanes 1 to 3 show the crude fractionation of 293T cell lysates (lane 1) expressing

    Article Snippet: Briefly, transfected 293T cells were collected 6 h posttransfection, washed three times with cold NTE buffer (150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), and suspended in 800 μl NTE buffer containing 6% (wt/vol) sucrose and a protease inhibitor cocktail (Sigma).

    Techniques: Mutagenesis, Fractionation, Expressing

    Design of the 5’ UTR library of naturally occurring and synthetic 5’ UTRs. RNA-seq and Ribo-seq datasets of HEK 293T, PC3, and human muscle cells, together with the GTEx database of human muscle tissue, were collected. Natural 5’UTRs with high TEs and low TEs in HEK 293T and RD cells, 5’ UTRs with various TEs in human muscle cells, and the 5’ UTRs with high mRNA counts in human muscle tissues were selected and added to the library. In addition, we designed synthetic 5’ UTRs by: i) collecting endogenous 5’ UTR sequences on the target cell type (HEK 293T, PC3 or human muscle cells) from public data; ii) extracting sequence features of the 5’ UTRs, including those nucleotides surrounding the AUG region; iii) training a Random Forest machine learning method for each cell type/tissue (HEK 293T, PC3 or human muscle cells), to learn a function that maps sequence features to mRNA expression levels and TEs; and iv) designing a set of 100 bp synthetic sequences that are predicted to maximize TEs and protein expression levels using genetic algorithms.

    Journal: bioRxiv

    Article Title: High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

    doi: 10.1101/2020.03.24.006486

    Figure Lengend Snippet: Design of the 5’ UTR library of naturally occurring and synthetic 5’ UTRs. RNA-seq and Ribo-seq datasets of HEK 293T, PC3, and human muscle cells, together with the GTEx database of human muscle tissue, were collected. Natural 5’UTRs with high TEs and low TEs in HEK 293T and RD cells, 5’ UTRs with various TEs in human muscle cells, and the 5’ UTRs with high mRNA counts in human muscle tissues were selected and added to the library. In addition, we designed synthetic 5’ UTRs by: i) collecting endogenous 5’ UTR sequences on the target cell type (HEK 293T, PC3 or human muscle cells) from public data; ii) extracting sequence features of the 5’ UTRs, including those nucleotides surrounding the AUG region; iii) training a Random Forest machine learning method for each cell type/tissue (HEK 293T, PC3 or human muscle cells), to learn a function that maps sequence features to mRNA expression levels and TEs; and iv) designing a set of 100 bp synthetic sequences that are predicted to maximize TEs and protein expression levels using genetic algorithms.

    Article Snippet: When HEK 293T cells were 80-90% confluent, cells were harvested with 0.25% trypsin for transfection.

    Techniques: RNA Sequencing Assay, Sequencing, Expressing

    Strategy for constructing HEK 293T cell lines with a landing pad and screening the 5’ UTR library using recombinase-based gene integration. A) Recombinase-based library screening workflow. B) Construction of the 5’ UTR library and schematic illustration of recombinase-based gene integration. C) We observed high reproducibility for barcode representations between two HEK-LP cell lines independently transfected with the library and a recombinase-expression plasmid; cells were sorted into three bins based on GFP expression (top 0-2.5%, top 2.5-5%, and top 5-10%). log2 values of normalized barcode counts are shown. R is the Pearson correlation coefficient.

    Journal: bioRxiv

    Article Title: High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

    doi: 10.1101/2020.03.24.006486

    Figure Lengend Snippet: Strategy for constructing HEK 293T cell lines with a landing pad and screening the 5’ UTR library using recombinase-based gene integration. A) Recombinase-based library screening workflow. B) Construction of the 5’ UTR library and schematic illustration of recombinase-based gene integration. C) We observed high reproducibility for barcode representations between two HEK-LP cell lines independently transfected with the library and a recombinase-expression plasmid; cells were sorted into three bins based on GFP expression (top 0-2.5%, top 2.5-5%, and top 5-10%). log2 values of normalized barcode counts are shown. R is the Pearson correlation coefficient.

    Article Snippet: When HEK 293T cells were 80-90% confluent, cells were harvested with 0.25% trypsin for transfection.

    Techniques: Library Screening, Transfection, Expressing, Plasmid Preparation

    Effects of combinatorial 5’ UTRs on GFP expression in various cell lines. A) We constructed six distinct 5’ UTR combinations by combining different pairwise permutations of the three validated 5’ UTR candidates with a CAACAA linker between them, and then inserted these combinations into the pVAX1-GFP plasmid directly upstream of the Kozak sequence. B) GFP expression from the 5’ UTR combinations on GFP expression in HEK 293T cells. C) Test of the single and combinatorial 5’ UTRs on GFP expression in various cell lines. The relative protein expression was normalized to that from the pVAX1-GFP plasmid, set as 1 and highlighted as a grey dotted line. Error bars indicate SD for three biological replicates. (*p

    Journal: bioRxiv

    Article Title: High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

    doi: 10.1101/2020.03.24.006486

    Figure Lengend Snippet: Effects of combinatorial 5’ UTRs on GFP expression in various cell lines. A) We constructed six distinct 5’ UTR combinations by combining different pairwise permutations of the three validated 5’ UTR candidates with a CAACAA linker between them, and then inserted these combinations into the pVAX1-GFP plasmid directly upstream of the Kozak sequence. B) GFP expression from the 5’ UTR combinations on GFP expression in HEK 293T cells. C) Test of the single and combinatorial 5’ UTRs on GFP expression in various cell lines. The relative protein expression was normalized to that from the pVAX1-GFP plasmid, set as 1 and highlighted as a grey dotted line. Error bars indicate SD for three biological replicates. (*p

    Article Snippet: When HEK 293T cells were 80-90% confluent, cells were harvested with 0.25% trypsin for transfection.

    Techniques: Expressing, Construct, Plasmid Preparation, Sequencing

    Schematic overview of recombinase-mediated 5’ UTR library screening strategy. Naturally occurring 5’ UTRs were extracted, analyzed, and used as the training set to generate synthetic 5’ UTRs for screening. Oligos encoding the 5’ UTR library were synthesized and cloned into plasmids containing a recombinase-recognition site and a GFP reporter. The resulting plasmids were transfected into the HEK 293T-LP cell line with the corresponding recombinase recognition site, resulting in targeted genomic insertion. The cells were sorted into bins based on GFP intensities, and the 5’ UTR sequences of each bin were amplified, sequenced, counted, and compared. The 5’ UTR candidates that enhanced GFP expression were selected and validated experimentally. Finally, the top-ranked validated 5’ UTRs were combined to test for increased gene expression.

    Journal: bioRxiv

    Article Title: High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

    doi: 10.1101/2020.03.24.006486

    Figure Lengend Snippet: Schematic overview of recombinase-mediated 5’ UTR library screening strategy. Naturally occurring 5’ UTRs were extracted, analyzed, and used as the training set to generate synthetic 5’ UTRs for screening. Oligos encoding the 5’ UTR library were synthesized and cloned into plasmids containing a recombinase-recognition site and a GFP reporter. The resulting plasmids were transfected into the HEK 293T-LP cell line with the corresponding recombinase recognition site, resulting in targeted genomic insertion. The cells were sorted into bins based on GFP intensities, and the 5’ UTR sequences of each bin were amplified, sequenced, counted, and compared. The 5’ UTR candidates that enhanced GFP expression were selected and validated experimentally. Finally, the top-ranked validated 5’ UTRs were combined to test for increased gene expression.

    Article Snippet: When HEK 293T cells were 80-90% confluent, cells were harvested with 0.25% trypsin for transfection.

    Techniques: Library Screening, Synthesized, Clone Assay, Transfection, Amplification, Expressing

    TROJAN associates with the ZMYND8 protein. ( A ) RIP assay and the subsequent qRT-PCR assay and semi–RT-PCR (inset). Relative quantification of TROJAN and MALAT1 expression in MDA-MB-231 LM2 RNA-protein complexes immunoprecipitated with IgG or ZMYND8 antibodies. MALAT1 was used as a negative control. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. ( B ) Confocal RNA fluorescence ISH and immunofluorescence images showing the colocalization of TROJAN and ZMYND8. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. ( C ) Schematic representation of Flag-tagged full-length human ZMYND8 and its deletion mutants. The ΔN, ΔInter, and ΔC constructs are depicted. The anti-Flag Western blot image showing ZMYND8 full-length or deletion mutant expression in human embryonic kidney (HEK) 293T cells. ( D ) RIP assay and the subsequent qRT-PCR assay performed in HEK293T cells ectopically expressing full-length Flag-tagged ZMYND8 and its deletion mutants. Relative quantification of TROJAN expression in RNA-protein complexes immunoprecipitated with IgG or Flag antibodies. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. ( E ) TROJAN motif and schematic representation of full-length TROJAN and its deletion mutants. ( F ) RIP assay and the subsequent qRT-PCR assay. HEK293T cells were cotransfected with full-length TROJAN (FL)/mutants (Δ1 to Δ4)/control vector as well as Flag-ZMYND8/Flag-vector (Flag-Vec). After 48 hours, the cell lysates were immunoprecipitated with beads coated with a Flag antibody. Two primers (#1 for FL, Δ1, and Δ4 and #2 for Δ2 and Δ3) were used in the qRT-PCR assay. Fold enrichment was adjusted by each input and Flag-Vec. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. * P

    Journal: Science Advances

    Article Title: The endogenous retrovirus-derived long noncoding RNA TROJAN promotes triple-negative breast cancer progression via ZMYND8 degradation

    doi: 10.1126/sciadv.aat9820

    Figure Lengend Snippet: TROJAN associates with the ZMYND8 protein. ( A ) RIP assay and the subsequent qRT-PCR assay and semi–RT-PCR (inset). Relative quantification of TROJAN and MALAT1 expression in MDA-MB-231 LM2 RNA-protein complexes immunoprecipitated with IgG or ZMYND8 antibodies. MALAT1 was used as a negative control. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. ( B ) Confocal RNA fluorescence ISH and immunofluorescence images showing the colocalization of TROJAN and ZMYND8. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. ( C ) Schematic representation of Flag-tagged full-length human ZMYND8 and its deletion mutants. The ΔN, ΔInter, and ΔC constructs are depicted. The anti-Flag Western blot image showing ZMYND8 full-length or deletion mutant expression in human embryonic kidney (HEK) 293T cells. ( D ) RIP assay and the subsequent qRT-PCR assay performed in HEK293T cells ectopically expressing full-length Flag-tagged ZMYND8 and its deletion mutants. Relative quantification of TROJAN expression in RNA-protein complexes immunoprecipitated with IgG or Flag antibodies. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. ( E ) TROJAN motif and schematic representation of full-length TROJAN and its deletion mutants. ( F ) RIP assay and the subsequent qRT-PCR assay. HEK293T cells were cotransfected with full-length TROJAN (FL)/mutants (Δ1 to Δ4)/control vector as well as Flag-ZMYND8/Flag-vector (Flag-Vec). After 48 hours, the cell lysates were immunoprecipitated with beads coated with a Flag antibody. Two primers (#1 for FL, Δ1, and Δ4 and #2 for Δ2 and Δ3) were used in the qRT-PCR assay. Fold enrichment was adjusted by each input and Flag-Vec. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. * P

    Article Snippet: Cell lines, transfection, and lentiviral shRNA vectors Immortalized HMEC and MCF-10A cells; human breast cancer cells MDA-MB-231, MDA-MB-468, Hs578t, and BT549; and human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection and cultured under standard conditions.

    Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing, Multiple Displacement Amplification, Immunoprecipitation, Negative Control, Two Tailed Test, Fluorescence, In Situ Hybridization, Immunofluorescence, Construct, Western Blot, Mutagenesis, Plasmid Preparation

    The F2 compound inhibits HIV-1 reverse transcriptase. A . Effects of F2 (5 μM) treatment on the synthesis of early and late viral DNA in human 293T cells challenged with the VSVg-pseudotyped HIV-1 vector, measured at 24 hours post-infection. AZT (5 μM) was used as a reference compound. The values represent amounts of DNA relative to control, untreated cell populations, with error bars showing standard deviations from three independent real-time quantitative PCR assays. B . Effect of compound F2 on HIV-1 RT activity in vitro , determined by measuring the [alpha32P]-dTTP incorporation. The experiment shown, performed with duplicate samples, is representative of two independent experiments. with error bars representing standard errors of the mean.

    Journal: Virology Journal

    Article Title: Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase

    doi: 10.1186/1743-422X-9-305

    Figure Lengend Snippet: The F2 compound inhibits HIV-1 reverse transcriptase. A . Effects of F2 (5 μM) treatment on the synthesis of early and late viral DNA in human 293T cells challenged with the VSVg-pseudotyped HIV-1 vector, measured at 24 hours post-infection. AZT (5 μM) was used as a reference compound. The values represent amounts of DNA relative to control, untreated cell populations, with error bars showing standard deviations from three independent real-time quantitative PCR assays. B . Effect of compound F2 on HIV-1 RT activity in vitro , determined by measuring the [alpha32P]-dTTP incorporation. The experiment shown, performed with duplicate samples, is representative of two independent experiments. with error bars representing standard errors of the mean.

    Article Snippet: Tissue culture-based infectivity assays Ten thousand human 293T cells were plated in 80 μl medium in each well of 96-well tissue culture plate.

    Techniques: Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Activity Assay, In Vitro

    The F2 compound blocks an early step of HIV-1 replication. A . Structure of compound F2 . B .Dose–response curve of F2 in 293T cells challenged with the VSVg pseudotyped pNL4-3lucR+E- vector. The effect of the compound on infection was determined by measurement of virus-encoded firefly luciferase activity. The experiment shown, performed with triplicate samples, is representative of six independent experiments. C . Dose–response curve of F2 in CEM-GFP cells challenged with the pLai3Luc2 HIV-1 vector [ 10 ].The numbers of GFP positive cells at 2 days post-infection were determined by flow cytometry and the experiment shown, performed with duplicate samples, is representative of two independent experiments. D . Dose–response curve of F2 in human PBMCs challenged with the NL4-3 Nef+ IRES rluc vector encoding renilla luciferase activity, measured at 5 days post-infection. The experiment shown was performed with eight replicate samples. The error bars (panels B-D) represent the standard errors of the mean.

    Journal: Virology Journal

    Article Title: Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase

    doi: 10.1186/1743-422X-9-305

    Figure Lengend Snippet: The F2 compound blocks an early step of HIV-1 replication. A . Structure of compound F2 . B .Dose–response curve of F2 in 293T cells challenged with the VSVg pseudotyped pNL4-3lucR+E- vector. The effect of the compound on infection was determined by measurement of virus-encoded firefly luciferase activity. The experiment shown, performed with triplicate samples, is representative of six independent experiments. C . Dose–response curve of F2 in CEM-GFP cells challenged with the pLai3Luc2 HIV-1 vector [ 10 ].The numbers of GFP positive cells at 2 days post-infection were determined by flow cytometry and the experiment shown, performed with duplicate samples, is representative of two independent experiments. D . Dose–response curve of F2 in human PBMCs challenged with the NL4-3 Nef+ IRES rluc vector encoding renilla luciferase activity, measured at 5 days post-infection. The experiment shown was performed with eight replicate samples. The error bars (panels B-D) represent the standard errors of the mean.

    Article Snippet: Tissue culture-based infectivity assays Ten thousand human 293T cells were plated in 80 μl medium in each well of 96-well tissue culture plate.

    Techniques: Plasmid Preparation, Infection, Luciferase, Activity Assay, Flow Cytometry, Cytometry

    The F2 compound is a NNRTI. A . Isobologram analysis of the effect of combining F2 with either AZT or NVP in human 293T cells challenged with the VSVg-pseudotyped HIV-1 vector. Depicted are isobologram plots for the 90% inhibitory level (EC 90 ). The dashed line indicates the values expected for an additive effect. Values below and to the left of the line indicate synergistic effects. FIC, fractional inhibitory concentration. B . Structure and synthesis of compound F2 compared to THR-50 [ 15 ]. Reagents: (a) 0.1 M in 1,2-dichloroethane – 2,2,2-trifluoroethanol (1:1), 5.0 mol% Sc(OTf) 3 , rt for 96h or microwaved at 140°C for 5min, > 90% yield; (b) 2,6-F 2 BzCl, THF/pyridine (5:1), rt for 2h, 55% yield; (c) Fe, AcOH, reflux, 97% yield; (d) 2,6-F 2 BnBr, NaH, THF, rt overnight, 75% yield.

    Journal: Virology Journal

    Article Title: Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase

    doi: 10.1186/1743-422X-9-305

    Figure Lengend Snippet: The F2 compound is a NNRTI. A . Isobologram analysis of the effect of combining F2 with either AZT or NVP in human 293T cells challenged with the VSVg-pseudotyped HIV-1 vector. Depicted are isobologram plots for the 90% inhibitory level (EC 90 ). The dashed line indicates the values expected for an additive effect. Values below and to the left of the line indicate synergistic effects. FIC, fractional inhibitory concentration. B . Structure and synthesis of compound F2 compared to THR-50 [ 15 ]. Reagents: (a) 0.1 M in 1,2-dichloroethane – 2,2,2-trifluoroethanol (1:1), 5.0 mol% Sc(OTf) 3 , rt for 96h or microwaved at 140°C for 5min, > 90% yield; (b) 2,6-F 2 BzCl, THF/pyridine (5:1), rt for 2h, 55% yield; (c) Fe, AcOH, reflux, 97% yield; (d) 2,6-F 2 BnBr, NaH, THF, rt overnight, 75% yield.

    Article Snippet: Tissue culture-based infectivity assays Ten thousand human 293T cells were plated in 80 μl medium in each well of 96-well tissue culture plate.

    Techniques: Plasmid Preparation, Concentration Assay, Reflux

    Identification of the AQP1 gene upstream promoter region. a , c and e Schematic representation of human AQP1 promoter reporter constructs. Fragments with various lengths of the AQP1 promoter region between − 4000 to 0 bp were cloned downstream of the firefly luciferase reporter. b , d and f Luciferase activity in HEK-293T cells transfected with firefly luciferase reporter plasmids containing various AQP1 upstream regions. The Renilla luciferase reporter was co-transfected with pGL3-basic or a plasmid reporter. The data represent the mean ± SEM. **P

    Journal: BMC Molecular Biology

    Article Title: pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

    doi: 10.1186/s12867-018-0104-9

    Figure Lengend Snippet: Identification of the AQP1 gene upstream promoter region. a , c and e Schematic representation of human AQP1 promoter reporter constructs. Fragments with various lengths of the AQP1 promoter region between − 4000 to 0 bp were cloned downstream of the firefly luciferase reporter. b , d and f Luciferase activity in HEK-293T cells transfected with firefly luciferase reporter plasmids containing various AQP1 upstream regions. The Renilla luciferase reporter was co-transfected with pGL3-basic or a plasmid reporter. The data represent the mean ± SEM. **P

    Article Snippet: The HEK-293T cell line was purchased from ATCC (American Type Culture Collection).

    Techniques: Construct, Clone Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    SPIB is a potential transcription factor capable of influencing pH-regulated AQP1 expression. a Putative SPIB binding site in the region between − 2300 to − 2200 bp upstream of AQP1. b Firefly luciferase activity normalized to Renilla luciferase activity in HEK-293T cells co-transfected with luciferase reporters with the wild-type or mutant AQP1 promoter after treatment at pH 7 or 8. c Chromatin immunoprecipitation (ChIP) in HEK293T cells, followed by real-time PCR amplification of the binding site within the AQP1 promoter region. d ChIP assays for the binding site in the AQP1 promoter were performed in HEK293T cells under different pH conditions. The data represent the mean ± SEM. ns no statistical significance; ***P

    Journal: BMC Molecular Biology

    Article Title: pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

    doi: 10.1186/s12867-018-0104-9

    Figure Lengend Snippet: SPIB is a potential transcription factor capable of influencing pH-regulated AQP1 expression. a Putative SPIB binding site in the region between − 2300 to − 2200 bp upstream of AQP1. b Firefly luciferase activity normalized to Renilla luciferase activity in HEK-293T cells co-transfected with luciferase reporters with the wild-type or mutant AQP1 promoter after treatment at pH 7 or 8. c Chromatin immunoprecipitation (ChIP) in HEK293T cells, followed by real-time PCR amplification of the binding site within the AQP1 promoter region. d ChIP assays for the binding site in the AQP1 promoter were performed in HEK293T cells under different pH conditions. The data represent the mean ± SEM. ns no statistical significance; ***P

    Article Snippet: The HEK-293T cell line was purchased from ATCC (American Type Culture Collection).

    Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification

    Transcription factors interacting with the enhancer region of the AQP1 gene. a Luciferase activity associated with the region between − 2.3k and 0 bp in AQP1 in HEK-293T cells transfected with small interfering RNAs (siRNAs) against EGR1, FOXL1, KLF5, NFIC, RFX5, SP1, THAP1, or SPIB or with a negative control (NC). b AQP1 mRNA levels in HEK-293T cells after SPIB knockdown and treatment at pH 7 and 8 were assessed with real-time PCR. The data represent the mean ± SEM. ns no statistical significance; ***P

    Journal: BMC Molecular Biology

    Article Title: pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

    doi: 10.1186/s12867-018-0104-9

    Figure Lengend Snippet: Transcription factors interacting with the enhancer region of the AQP1 gene. a Luciferase activity associated with the region between − 2.3k and 0 bp in AQP1 in HEK-293T cells transfected with small interfering RNAs (siRNAs) against EGR1, FOXL1, KLF5, NFIC, RFX5, SP1, THAP1, or SPIB or with a negative control (NC). b AQP1 mRNA levels in HEK-293T cells after SPIB knockdown and treatment at pH 7 and 8 were assessed with real-time PCR. The data represent the mean ± SEM. ns no statistical significance; ***P

    Article Snippet: The HEK-293T cell line was purchased from ATCC (American Type Culture Collection).

    Techniques: Luciferase, Activity Assay, Transfection, Negative Control, Real-time Polymerase Chain Reaction

    Increased pH elevates AQP1 expression. a AQP1 mRNA levels in HEK-293T cells with various pH treatments. b Luciferase activity in HEK-293T cells transfected with firefly luciferase reporter plasmids containing AQP1 upstream regions. The Renilla luciferase reporter was co-transfected with pGL3-basic or a plasmid reporter. The data represent the mean ± SEM. ns no statistical significance; *P

    Journal: BMC Molecular Biology

    Article Title: pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

    doi: 10.1186/s12867-018-0104-9

    Figure Lengend Snippet: Increased pH elevates AQP1 expression. a AQP1 mRNA levels in HEK-293T cells with various pH treatments. b Luciferase activity in HEK-293T cells transfected with firefly luciferase reporter plasmids containing AQP1 upstream regions. The Renilla luciferase reporter was co-transfected with pGL3-basic or a plasmid reporter. The data represent the mean ± SEM. ns no statistical significance; *P

    Article Snippet: The HEK-293T cell line was purchased from ATCC (American Type Culture Collection).

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

    AQP1 is regulated by SPIB. a Luciferase activity associated with the region between −2300 and −2200 bp in HEK-293T cells transfected with SPIB. b AQP1 mRNA levels in HEK-293T cells transfected with SPIB. The data represent the mean ± SEM. ns no statistical significance; ***P

    Journal: BMC Molecular Biology

    Article Title: pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

    doi: 10.1186/s12867-018-0104-9

    Figure Lengend Snippet: AQP1 is regulated by SPIB. a Luciferase activity associated with the region between −2300 and −2200 bp in HEK-293T cells transfected with SPIB. b AQP1 mRNA levels in HEK-293T cells transfected with SPIB. The data represent the mean ± SEM. ns no statistical significance; ***P

    Article Snippet: The HEK-293T cell line was purchased from ATCC (American Type Culture Collection).

    Techniques: Luciferase, Activity Assay, Transfection

    ExomiR-155 mediates the adipocyte metabolism by downregulating PPARγ. The adipocytes in 50 μg of exosomes purified from cancer-associated conditioned medium (CA-CM). ( a ) Exosomes originating from CA-CM viewed by electron microscopy (scale bar, 200 nm). ( b ) Exosomes from CA-CM were analysed by western blot. ( c ) NanoSight analysis of exosomes derived from CA-CM. ( d ) 4 T-1 were cocultivated in the presence or absence of adipocytes. After 3 days, exosomal miRNAs were further verified by qPCR. And RNA was extracted from the adipocytes and subjected to qPCR analysis with primers specific to mature miRNA. ( e ) The predicted miR-155 binding site in the 3’UTR of the PPARγ gene from TargetScan. ( f ) The GV272 vector containing the 3’UTR of the target gene harbouring wild-type (wt) or mutated (mt) miRNA binding sites was transfected into HEK 293 T cells stably expressing miRNA or empty vector (as a control). Luciferase activity was analysed at 48 h post-transfection, and the ratio of firefly luciferase activity to Renilla luciferase activity is shown. ( g ) Breast cancer cells were transfected with miRNA-155 pre-miRNA or miR-155 inhibitor, mature adipocytes were transfected with miR-155 mimic as the positive control and the control vector was applied as the negative control. Mature adipocytes cultured in the presence or absence of tumour exosomes for 3 days were stained with red oil, and Western blot analysis of related protein expression in different groups ( h ). Data are presented as the mean ± S.D. of at least three independent experiments. *  P

    Journal: Molecular Cancer

    Article Title: Tumour-originated exosomal miR-155 triggers cancer-associated cachexia to promote tumour progression

    doi: 10.1186/s12943-018-0899-5

    Figure Lengend Snippet: ExomiR-155 mediates the adipocyte metabolism by downregulating PPARγ. The adipocytes in 50 μg of exosomes purified from cancer-associated conditioned medium (CA-CM). ( a ) Exosomes originating from CA-CM viewed by electron microscopy (scale bar, 200 nm). ( b ) Exosomes from CA-CM were analysed by western blot. ( c ) NanoSight analysis of exosomes derived from CA-CM. ( d ) 4 T-1 were cocultivated in the presence or absence of adipocytes. After 3 days, exosomal miRNAs were further verified by qPCR. And RNA was extracted from the adipocytes and subjected to qPCR analysis with primers specific to mature miRNA. ( e ) The predicted miR-155 binding site in the 3’UTR of the PPARγ gene from TargetScan. ( f ) The GV272 vector containing the 3’UTR of the target gene harbouring wild-type (wt) or mutated (mt) miRNA binding sites was transfected into HEK 293 T cells stably expressing miRNA or empty vector (as a control). Luciferase activity was analysed at 48 h post-transfection, and the ratio of firefly luciferase activity to Renilla luciferase activity is shown. ( g ) Breast cancer cells were transfected with miRNA-155 pre-miRNA or miR-155 inhibitor, mature adipocytes were transfected with miR-155 mimic as the positive control and the control vector was applied as the negative control. Mature adipocytes cultured in the presence or absence of tumour exosomes for 3 days were stained with red oil, and Western blot analysis of related protein expression in different groups ( h ). Data are presented as the mean ± S.D. of at least three independent experiments. * P

    Article Snippet: The mouse breast cancer cell lines 4 T-1, C2C12 and HEK 293 T cells were obtained from American Type Culture Collection (ATCC, Shanghai) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% exosome-free foetal bovine serum (FBS, Shin Chin Industrial, SCI) and 1% penicillin–streptomycin (HyClone, Logan, UT, USA) in a humidified 37 °C incubator with 5% CO2 .

    Techniques: Purification, Electron Microscopy, Western Blot, Derivative Assay, Real-time Polymerase Chain Reaction, Binding Assay, Plasmid Preparation, Transfection, Stable Transfection, Expressing, Luciferase, Activity Assay, Positive Control, Negative Control, Cell Culture, Staining

    Membrane localization of EGFP-tagged Ca V 1.2e1b and Ca V 1.2e1c channels when expressed with α 2 δ or with α 2 δ + β 1b subunits in HEK 293 cells

    Journal:

    Article Title: A Novel CaV1.2 N Terminus Expressed in Smooth Muscle Cells of Resistance Size Arteries Modifies Channel Regulation by Auxiliary Subunits *1.2 N Terminus Expressed in Smooth Muscle Cells of Resistance Size Arteries Modifies Channel Regulation by Auxiliary Subunits * S

    doi: 10.1074/jbc.M610623200

    Figure Lengend Snippet: Membrane localization of EGFP-tagged Ca V 1.2e1b and Ca V 1.2e1c channels when expressed with α 2 δ or with α 2 δ + β 1b subunits in HEK 293 cells

    Article Snippet: HEK 293 cells were transfected with different combinations of pEGFP-CaV 1.2e1b-N3 or pEGFP-CaV 1.2e1c-N3 (0.5 μ g) and pcDNA3- α 2 δ -1 and pGW- β 1b (1 μ g of each).

    Techniques:

    Analysis of E1E2 of diverse HCV genotypes. (A) A series of threefold dilutions of lysates of HEK 293T cells cotransfected with the MLV packaging/transfer vector and a plasmid expressing E1E2 of different HCV genotypes were analyzed for the presence of

    Journal:

    Article Title: Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein

    doi: 10.1128/JVI.79.17.11095-11104.2005

    Figure Lengend Snippet: Analysis of E1E2 of diverse HCV genotypes. (A) A series of threefold dilutions of lysates of HEK 293T cells cotransfected with the MLV packaging/transfer vector and a plasmid expressing E1E2 of different HCV genotypes were analyzed for the presence of

    Article Snippet: Human Huh-7 hepatoma cells ( ) and human epithelial kidney (HEK) 293T cells (ATCC CRL-1573) were propagated as described ( ).

    Techniques: Plasmid Preparation, Expressing

    NS1-specific antibodies activate Fc-FcR effector functions in vitro. To examine the ability of NS1-specific antibodies to activate Fc-FcR mediated effector functions, a , b Vero cells were infected with MR766 and PRVABC59 Zika viruses or c , d HEK 293T cells were transfected with NS1 from MR766 and PRVABC59 Zika viruses. Infected Vero cells or transfected HEK 293T cells were used as targets for measuring antibody-mediated effector functions with a genetically modified Jurkat cell line expressing the human FcRγIIIa with an inducible luciferase reporter gene. Fold induction was measured in relative light units and calculated by subtracting background signal from wells without effector cells then dividing wells with antibody by wells with no antibody added. All mAbs were tested at a starting concentration of 10 μg per mL and were serially diluted four-fold. Assays were performed twice as technical duplicates and one of two replicates is shown. A non-linear regression best-fit curve was generated for each dataset using GraphPad Prism 5. Error bars represent SEM

    Journal: Nature Communications

    Article Title: Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model

    doi: 10.1038/s41467-018-07008-0

    Figure Lengend Snippet: NS1-specific antibodies activate Fc-FcR effector functions in vitro. To examine the ability of NS1-specific antibodies to activate Fc-FcR mediated effector functions, a , b Vero cells were infected with MR766 and PRVABC59 Zika viruses or c , d HEK 293T cells were transfected with NS1 from MR766 and PRVABC59 Zika viruses. Infected Vero cells or transfected HEK 293T cells were used as targets for measuring antibody-mediated effector functions with a genetically modified Jurkat cell line expressing the human FcRγIIIa with an inducible luciferase reporter gene. Fold induction was measured in relative light units and calculated by subtracting background signal from wells without effector cells then dividing wells with antibody by wells with no antibody added. All mAbs were tested at a starting concentration of 10 μg per mL and were serially diluted four-fold. Assays were performed twice as technical duplicates and one of two replicates is shown. A non-linear regression best-fit curve was generated for each dataset using GraphPad Prism 5. Error bars represent SEM

    Article Snippet: Cells and viruses Human embryonic kidney (HEK) 293T cells (American Type Culture Collection; ATCC Cat. No. CRL-1573) and African green monkey kidney cells (Vero; ATCC Cat. No. CCL-81) were grown in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics (100 units per mL penicillin–100 µg per mL streptomycin [Pen-Strep]; Gibco).

    Techniques: In Vitro, Infection, Transfection, Genetically Modified, Expressing, Luciferase, Concentration Assay, Generated

    A , The Stin2 VNTR differentially represses transcription in reporter gene assays. JAr or 293T cells were transiently transfected with the constructs including VNTR allelic variants (Stin2.9, Stin2.10, and Stin2.12) fused to Luciferase gene, harvested,

    Journal: The Journal of Neuroscience

    Article Title: Differential Regulation of the Serotonin Transporter Gene by Lithium Is Mediated by Transcription Factors, CCCTC Binding Protein and Y-Box Binding Protein 1, through the Polymorphic Intron 2 Variable Number Tandem Repeat

    doi: 10.1523/JNEUROSCI.0892-06.2007

    Figure Lengend Snippet: A , The Stin2 VNTR differentially represses transcription in reporter gene assays. JAr or 293T cells were transiently transfected with the constructs including VNTR allelic variants (Stin2.9, Stin2.10, and Stin2.12) fused to Luciferase gene, harvested,

    Article Snippet: Transformed human epithelial kidney cells (293T) (ATCC CRL-1573; American Type Culture Collection) were maintained in DMEM supplemented with 10% fetal calf serum and 50 μg/ml gentamycin.

    Techniques: Transfection, Construct, Luciferase

    Position L665 in HCV E2 is highly sensitive to amino-acid substitutions. (A) The leucine at position 665 in H77/JFH1 was substituted for the indicated amino acids, and Huh7.5 cells were transfected with in vitro transcribed RNA of recombinants harboring these substitutions. Supernatants were collected at the indicated time-points and HCV infectivity titers were determined as described in Materials and Methods. (B) 293T cells were transiently transfected with vectors expressing L665 mutants of H77 E1E2 in cis. E1/E2 protein complexes were gently released using 1% nDDM detergent with protease inhibitors and benzonase. The samples were subjected to AR3A (conformational E2-specific antibody) immunoprecipitation and SDS-PAGE and western blot transfer was performed on the enriched fractions. E2 was detected using antibody H52 and E1 was detected using antibody A4.

    Journal: PLoS Pathogens

    Article Title: Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

    doi: 10.1371/journal.ppat.1006214

    Figure Lengend Snippet: Position L665 in HCV E2 is highly sensitive to amino-acid substitutions. (A) The leucine at position 665 in H77/JFH1 was substituted for the indicated amino acids, and Huh7.5 cells were transfected with in vitro transcribed RNA of recombinants harboring these substitutions. Supernatants were collected at the indicated time-points and HCV infectivity titers were determined as described in Materials and Methods. (B) 293T cells were transiently transfected with vectors expressing L665 mutants of H77 E1E2 in cis. E1/E2 protein complexes were gently released using 1% nDDM detergent with protease inhibitors and benzonase. The samples were subjected to AR3A (conformational E2-specific antibody) immunoprecipitation and SDS-PAGE and western blot transfer was performed on the enriched fractions. E2 was detected using antibody H52 and E1 was detected using antibody A4.

    Article Snippet: E1/E2 interaction assay 293T cells (American Type Culture Collection, ID: CRL-1573) were plated at 400.000 cells/well in a 6-well plate and the following day the cells were transfected using Lipofectamine 2000 (Invitrogen) with 5 μg of E1E2 expression plasmids.

    Techniques: Transfection, In Vitro, Infection, Expressing, Immunoprecipitation, SDS Page, Western Blot

    Sp1 binds to the human Klotho gene ( KL ) promoter. ( a ) HEK-293 cells were co-transfected with Full KL/LUC reporter and pcDNA3 or pcDNA3-Sp1 plasmid. 24 h after transfection, cells were lysed and transcriptional activity was detected by dual-luciferase assays. ( b ) Cells were transfected with full length or truncated reporter, combined with pcDNA3 or pcDNA3-Sp1 plasmid. 24 h after transfection, HEK-293 cells were lysed and transcriptional activity was detected. ( c ) HEK-293 cells were transfected with KL5/LUC or mutated KL5/LUC reporter, and transcriptional activity was detected as before. As an internal control, Rinella (pRL-TK) was used to normalize the transfection efficiency. Mean ± SD of triplicate experiments performed independently. *** p

    Journal: BMC Molecular and Cell Biology

    Article Title: Klotho is regulated by transcription factor Sp1 in renal tubular epithelial cells

    doi: 10.1186/s12860-020-00292-z

    Figure Lengend Snippet: Sp1 binds to the human Klotho gene ( KL ) promoter. ( a ) HEK-293 cells were co-transfected with Full KL/LUC reporter and pcDNA3 or pcDNA3-Sp1 plasmid. 24 h after transfection, cells were lysed and transcriptional activity was detected by dual-luciferase assays. ( b ) Cells were transfected with full length or truncated reporter, combined with pcDNA3 or pcDNA3-Sp1 plasmid. 24 h after transfection, HEK-293 cells were lysed and transcriptional activity was detected. ( c ) HEK-293 cells were transfected with KL5/LUC or mutated KL5/LUC reporter, and transcriptional activity was detected as before. As an internal control, Rinella (pRL-TK) was used to normalize the transfection efficiency. Mean ± SD of triplicate experiments performed independently. *** p

    Article Snippet: Western blot analysisHuman HK-2 cells and human HEK-293 cells were lysed and total protein separated by western blot as described previously.

    Techniques: Transfection, Plasmid Preparation, Activity Assay, Luciferase

    Overexpression of Sp1 upregulates Klotho expression. HK-2 cells ( a - b ) or HEK-293 cells ( c - d ) were transfected with increasing amount of pcDNA3-Sp1 plasmid or empty control. 48 h after transfection, total proteins were isolated and the protein levels of Sp1 and Klotho were detected by Western blots ( a / c ). Total cell RNAs were isolated for quantitative RT-PCR to analyze the mRNA level of Klotho ( b / d ). Data were expressed as the mean ± SD in three independent experiments. *** p

    Journal: BMC Molecular and Cell Biology

    Article Title: Klotho is regulated by transcription factor Sp1 in renal tubular epithelial cells

    doi: 10.1186/s12860-020-00292-z

    Figure Lengend Snippet: Overexpression of Sp1 upregulates Klotho expression. HK-2 cells ( a - b ) or HEK-293 cells ( c - d ) were transfected with increasing amount of pcDNA3-Sp1 plasmid or empty control. 48 h after transfection, total proteins were isolated and the protein levels of Sp1 and Klotho were detected by Western blots ( a / c ). Total cell RNAs were isolated for quantitative RT-PCR to analyze the mRNA level of Klotho ( b / d ). Data were expressed as the mean ± SD in three independent experiments. *** p

    Article Snippet: Western blot analysisHuman HK-2 cells and human HEK-293 cells were lysed and total protein separated by western blot as described previously.

    Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Isolation, Western Blot, Quantitative RT-PCR

    Knockdown of Sp1 reduces klotho expression and induces α-SMA and Fibronectin expression. HK-2 cells ( a - b ) or HEK-293 cells ( c - d ) were transfected with increasing amount of Sp1-targeted siRNA or control siRNA. 48 h after transfection, total proteins were isolated and the protein levels of Sp1 and Klotho were detected by Western blots ( a / c ). Cell total RNAs were isolated for quantitative RT-PCR to analyze the mRNA level of Klotho ( b / d ). Total proteins were isolated and the protein levels of E-cadherin, α-SMA, Fibronectin were detected by Western blots (e). Data were expressed as the mean ± SD of three independent experiments. *** p

    Journal: BMC Molecular and Cell Biology

    Article Title: Klotho is regulated by transcription factor Sp1 in renal tubular epithelial cells

    doi: 10.1186/s12860-020-00292-z

    Figure Lengend Snippet: Knockdown of Sp1 reduces klotho expression and induces α-SMA and Fibronectin expression. HK-2 cells ( a - b ) or HEK-293 cells ( c - d ) were transfected with increasing amount of Sp1-targeted siRNA or control siRNA. 48 h after transfection, total proteins were isolated and the protein levels of Sp1 and Klotho were detected by Western blots ( a / c ). Cell total RNAs were isolated for quantitative RT-PCR to analyze the mRNA level of Klotho ( b / d ). Total proteins were isolated and the protein levels of E-cadherin, α-SMA, Fibronectin were detected by Western blots (e). Data were expressed as the mean ± SD of three independent experiments. *** p

    Article Snippet: Western blot analysisHuman HK-2 cells and human HEK-293 cells were lysed and total protein separated by western blot as described previously.

    Techniques: Expressing, Transfection, Isolation, Western Blot, Quantitative RT-PCR

    PMTb-GFP initially colocalizes with CTxB-594, but then diverges and is trafficked to and translocated from acidic endosomes. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q  as described in Methods. Seven h post-transfection cells were treated with ( a ) NH 4 Cl or ( b ) bafilomycin A1 (BAF) at the indicated concentrations for 15 min before treatment with 100 ng/mL rPMT. After 15 h incubation, cells were harvested and assayed for SRE reporter gene activity, as described in the Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT-treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of three experiments ± S.D. with each experiment performed in triplicate, where *  p

    Journal: Toxins

    Article Title: Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    doi: 10.3390/toxins3030218

    Figure Lengend Snippet: PMTb-GFP initially colocalizes with CTxB-594, but then diverges and is trafficked to and translocated from acidic endosomes. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q as described in Methods. Seven h post-transfection cells were treated with ( a ) NH 4 Cl or ( b ) bafilomycin A1 (BAF) at the indicated concentrations for 15 min before treatment with 100 ng/mL rPMT. After 15 h incubation, cells were harvested and assayed for SRE reporter gene activity, as described in the Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT-treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of three experiments ± S.D. with each experiment performed in triplicate, where * p

    Article Snippet: Cell Culture HEK 293-T cells (ATCC # CRL-11268) and Swiss 3T3 cells (ATCC # CCL-92) were cultured and maintained at 37 °C and 5% CO2 in DMEM (Gibco) with 10% fetal bovine serum (FBS), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Transfection, Luciferase, Incubation, Activity Assay, Activation Assay

    Arf6-dependent internalization and trafficking of PMT. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids, pcDNA3-Gα q , and plasmids containing Arf6 or Arf6 mutants, as described in Methods. Seven hours post-transfection cells were treated with 100 ng/mL PMT and incubated for 15 h before assaying for SRE reporter gene activity. SRE fold activation was determined by dividing SRE reporter gene activity in PMT treated cells by SRE reporter gene activity in untreated control cells. Fold activation was then normalized to empty vector control (pcDNA3). Data is expressed as an average of eight experiments ± S.D., with each experiment performed in triplicate, where *  p

    Journal: Toxins

    Article Title: Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    doi: 10.3390/toxins3030218

    Figure Lengend Snippet: Arf6-dependent internalization and trafficking of PMT. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids, pcDNA3-Gα q , and plasmids containing Arf6 or Arf6 mutants, as described in Methods. Seven hours post-transfection cells were treated with 100 ng/mL PMT and incubated for 15 h before assaying for SRE reporter gene activity. SRE fold activation was determined by dividing SRE reporter gene activity in PMT treated cells by SRE reporter gene activity in untreated control cells. Fold activation was then normalized to empty vector control (pcDNA3). Data is expressed as an average of eight experiments ± S.D., with each experiment performed in triplicate, where * p

    Article Snippet: Cell Culture HEK 293-T cells (ATCC # CRL-11268) and Swiss 3T3 cells (ATCC # CCL-92) were cultured and maintained at 37 °C and 5% CO2 in DMEM (Gibco) with 10% fetal bovine serum (FBS), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Transfection, Luciferase, Incubation, Activity Assay, Activation Assay, Plasmid Preparation

    PMTb-GFP initially colocalizes with Tfn-Texas Red, but then diverges and is trafficked to endosomes through a PI3-kinase-independent process. Shown are confocal microscope images of Swiss 3T3 cells after treatment with PMTb-GFP, with the endosomes marked by Transferrin-Texas Red (Tfn-TR). Cells were treated with 260 µg/mL PMTb-GFP and with 20 µg/mL Tfn-TR for 3.5 h to visualize the endosomes. Cells were visualized by confocal microscopy using a 40× objective. ( a ) PMTb-GFP; ( b ) Tfn-TR; ( c ) merged image of (a) and (b); ( d ) corresponding phase-contrast image. Inset: Enlargement of the indicated section of the image, showing co-localization of PMTb-GFP and Tfn-TR; ( e ) HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q  as described in Methods. Seven h post-transfection cells were treated with LY294002 at the indicated concentrations and incubated for 15 min before treatment with 100 ng/mL PMT. After 15 h incubation, cells were harvested and assayed for SRE reporter gene activity, as described in Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT-treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of three experiments ± S.D., with each experiment performed in triplicate, where **  p

    Journal: Toxins

    Article Title: Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    doi: 10.3390/toxins3030218

    Figure Lengend Snippet: PMTb-GFP initially colocalizes with Tfn-Texas Red, but then diverges and is trafficked to endosomes through a PI3-kinase-independent process. Shown are confocal microscope images of Swiss 3T3 cells after treatment with PMTb-GFP, with the endosomes marked by Transferrin-Texas Red (Tfn-TR). Cells were treated with 260 µg/mL PMTb-GFP and with 20 µg/mL Tfn-TR for 3.5 h to visualize the endosomes. Cells were visualized by confocal microscopy using a 40× objective. ( a ) PMTb-GFP; ( b ) Tfn-TR; ( c ) merged image of (a) and (b); ( d ) corresponding phase-contrast image. Inset: Enlargement of the indicated section of the image, showing co-localization of PMTb-GFP and Tfn-TR; ( e ) HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q as described in Methods. Seven h post-transfection cells were treated with LY294002 at the indicated concentrations and incubated for 15 min before treatment with 100 ng/mL PMT. After 15 h incubation, cells were harvested and assayed for SRE reporter gene activity, as described in Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT-treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of three experiments ± S.D., with each experiment performed in triplicate, where ** p

    Article Snippet: Cell Culture HEK 293-T cells (ATCC # CRL-11268) and Swiss 3T3 cells (ATCC # CCL-92) were cultured and maintained at 37 °C and 5% CO2 in DMEM (Gibco) with 10% fetal bovine serum (FBS), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Microscopy, Confocal Microscopy, Transfection, Luciferase, Incubation, Activity Assay, Activation Assay

    Both actin and microtubule dynamics are important for PMT trafficking to translocation-productive acidic late endosomes. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q  as described in Methods. Seven h post-transfection cells were treated with cytochalasin D ( a ) or nocodazole ( b ) at the indicated concentrations for 15 min before treatment with 100 ng/mL PMT. After 15 h incubation, cells were assayed for SRE reporter gene activity, as described in Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT-treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of three experiments ± S.D., with each experiment performed in triplicate, where *  p

    Journal: Toxins

    Article Title: Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    doi: 10.3390/toxins3030218

    Figure Lengend Snippet: Both actin and microtubule dynamics are important for PMT trafficking to translocation-productive acidic late endosomes. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q as described in Methods. Seven h post-transfection cells were treated with cytochalasin D ( a ) or nocodazole ( b ) at the indicated concentrations for 15 min before treatment with 100 ng/mL PMT. After 15 h incubation, cells were assayed for SRE reporter gene activity, as described in Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT-treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of three experiments ± S.D., with each experiment performed in triplicate, where * p

    Article Snippet: Cell Culture HEK 293-T cells (ATCC # CRL-11268) and Swiss 3T3 cells (ATCC # CCL-92) were cultured and maintained at 37 °C and 5% CO2 in DMEM (Gibco) with 10% fetal bovine serum (FBS), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Translocation Assay, Transfection, Luciferase, Incubation, Activity Assay, Activation Assay

    Treatment with brefeldin A enhances PMT activity. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q  as described in Methods. Seven h post-transfection cells were treated with brefeldin A (BFA) at the indicated concentrations ( a ) or with a combination of BFA (1 µM) and NH 4 Cl at the indicated concentrations ( b ) for 15 min before treatment with 100 ng/mL PMT. After 15 h incubation, the cells were assayed for SRE reporter gene activity as described in Methods. ( c ) HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids, pcDNA3-Gα q , and plasmids containing Arf6 Q67L or Arf6 T157A, as described in Methods. Seven h post-transfection cells were treated with BFA (1 µM) for 15 min before treatment with 100 ng/mL PMT. After 15 h incubation, cells were assayed for SRE reporter gene activity as described in Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of two experiments ± S.D., with each performed in triplicate, where *  p

    Journal: Toxins

    Article Title: Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    doi: 10.3390/toxins3030218

    Figure Lengend Snippet: Treatment with brefeldin A enhances PMT activity. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q as described in Methods. Seven h post-transfection cells were treated with brefeldin A (BFA) at the indicated concentrations ( a ) or with a combination of BFA (1 µM) and NH 4 Cl at the indicated concentrations ( b ) for 15 min before treatment with 100 ng/mL PMT. After 15 h incubation, the cells were assayed for SRE reporter gene activity as described in Methods. ( c ) HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids, pcDNA3-Gα q , and plasmids containing Arf6 Q67L or Arf6 T157A, as described in Methods. Seven h post-transfection cells were treated with BFA (1 µM) for 15 min before treatment with 100 ng/mL PMT. After 15 h incubation, cells were assayed for SRE reporter gene activity as described in Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of two experiments ± S.D., with each performed in triplicate, where * p

    Article Snippet: Cell Culture HEK 293-T cells (ATCC # CRL-11268) and Swiss 3T3 cells (ATCC # CCL-92) were cultured and maintained at 37 °C and 5% CO2 in DMEM (Gibco) with 10% fetal bovine serum (FBS), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Activity Assay, Transfection, Luciferase, Incubation, Activation Assay

    Examination of the internalization and β-arrestin-binding properties of N321K-V2R. HEK293 cells were transiently transfected with the plasmids of the indicated BRET partners and after 24 hours, the cells were exposed to AVP or vehicle (veh). A

    Journal: Molecular Endocrinology

    Article Title: Altered Agonist Sensitivity of a Mutant V2 Receptor Suggests a Novel Therapeutic Strategy for Nephrogenic Diabetes Insipidus

    doi: 10.1210/me.2013-1424

    Figure Lengend Snippet: Examination of the internalization and β-arrestin-binding properties of N321K-V2R. HEK293 cells were transiently transfected with the plasmids of the indicated BRET partners and after 24 hours, the cells were exposed to AVP or vehicle (veh). A

    Article Snippet: The human embryonic kidney (HEK293) cells were from ATCC.

    Techniques: Binding Assay, Transfection, Bioluminescence Resonance Energy Transfer

    Measurement of the cAMP signal upon different agonist stimuli. HEK293 cells were transiently transfected with the WT-V2R or the N321K-V2R and the Epac-BRET sensor. After 24 hours, the BRET measurements were implemented. A–D, Dose-response curves

    Journal: Molecular Endocrinology

    Article Title: Altered Agonist Sensitivity of a Mutant V2 Receptor Suggests a Novel Therapeutic Strategy for Nephrogenic Diabetes Insipidus

    doi: 10.1210/me.2013-1424

    Figure Lengend Snippet: Measurement of the cAMP signal upon different agonist stimuli. HEK293 cells were transiently transfected with the WT-V2R or the N321K-V2R and the Epac-BRET sensor. After 24 hours, the BRET measurements were implemented. A–D, Dose-response curves

    Article Snippet: The human embryonic kidney (HEK293) cells were from ATCC.

    Techniques: Transfection, Bioluminescence Resonance Energy Transfer

    Measurement of the cAMP signal upon AVP and dDAVP stimuli. HEK293 cells were transiently transfected with the WT-V2R or the N321K-V2R and the Epac-BRET sensor. After 24 hours, the BRET measurements were implemented. A, Cells were stimulated with 10 nM

    Journal: Molecular Endocrinology

    Article Title: Altered Agonist Sensitivity of a Mutant V2 Receptor Suggests a Novel Therapeutic Strategy for Nephrogenic Diabetes Insipidus

    doi: 10.1210/me.2013-1424

    Figure Lengend Snippet: Measurement of the cAMP signal upon AVP and dDAVP stimuli. HEK293 cells were transiently transfected with the WT-V2R or the N321K-V2R and the Epac-BRET sensor. After 24 hours, the BRET measurements were implemented. A, Cells were stimulated with 10 nM

    Article Snippet: The human embryonic kidney (HEK293) cells were from ATCC.

    Techniques: Transfection, Bioluminescence Resonance Energy Transfer

    Examination of the cell surface expression of the N321K-V2R. Immunofluorescence microscopy analysis of HEK293 cells transiently expressing WT (A and C) or N321K (B and D) HA-tagged V2R. The samples were stained with anti-HA-Alexa 488 mouse monoclonal

    Journal: Molecular Endocrinology

    Article Title: Altered Agonist Sensitivity of a Mutant V2 Receptor Suggests a Novel Therapeutic Strategy for Nephrogenic Diabetes Insipidus

    doi: 10.1210/me.2013-1424

    Figure Lengend Snippet: Examination of the cell surface expression of the N321K-V2R. Immunofluorescence microscopy analysis of HEK293 cells transiently expressing WT (A and C) or N321K (B and D) HA-tagged V2R. The samples were stained with anti-HA-Alexa 488 mouse monoclonal

    Article Snippet: The human embryonic kidney (HEK293) cells were from ATCC.

    Techniques: Expressing, Immunofluorescence, Microscopy, Staining