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  • 99
    ATCC hek293 cells
    Lysate-based CETSA for SLC1A2 transiently expressed in <t>HEK293</t> cells. (a) Chemical structure of WAY-213613. (b) The results of immunoblotting show the thermostability of FLAG-SLC1A2 following heating in the presence (+) or absence (−) of WAY-213613 (100 μM). For melting curve analysis, the signal intensity of the thermostable protein was normalized to the intensity of the 45 °C sample. Data are means ± the standard deviation of biological triplicate measurements. The immunoblots are representative results of three biological replicates. Tubulin was analyzed as a control. Legend: L.E., long exposure; WAY, WAY-213613.
    Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 15060 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore hek293 cells
    Disease-associated mutations increase temperature activation of TRPM3. <t>HEK293</t> cells were transfected with the human TRPM3α2, or its mutants; fura-2 Ca 2+ imaging experiments were performed as described in the Materials and methods section. ( A-C ) Averaged fluorescence ratio traces (340 nm/380 nm) for all PregS-responsive cells from one coverslip in cells expressing TRPM3 ( A ), V992M ( B ) and P1092Q ( C ). The cells were first stimulated by increasing the temperature to 37°C, followed by 10 μM primidone at room temperature to facilitate return of Ca 2+ to baseline, finally 25 μM PregS was applied. ( D ) The change in 340/380 ratio induced by 37°C expressed as a fraction of the response to 25 μM PregS. ( E ) 340/380 ratios at baseline, in response to 37°C and in response to 25 μM PregS. Each symbol represents the average value from all PregS-responsive cells from one coverslip, lines connect data points from the same coverslip. Each coverslips had around 70–80 PregS-responsive cells in the WT group; 35–45 for P1092Q, and 15–25 for V992M. Data were collected from three independent transfections.
    Hek293 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hek 293 cells
    Detection of selectin receptors with antibodies and fusion proteins on <t>HEK-293</t> EBOV-GP cells (6a) Flow cytometry analysis showing the binding of different monoclonal antibodies against CD44v6, CD44s, DR3, CD24, HLAabc, and NCAM (CD56) to HEK-293 EBOV-GP cells compared to vector transfected controls. (6b), Binding of NKG2D-Fc to its ligands MICA/B (MHC I chain related molecule) is reduced in HEK-293 transfected cells either with the Marburg-GP or the 7916-5 GP, while binding of CD24 and CD44s is significantly increased. The expression of HLA is reduced. The binding of mAb against CD44v6, DR3 is not significantly changed.
    Hek 293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12989 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hek293 cells
    Immunostaining of <t>HEK293</t> cells stably expressing EGFP-PICK1 alone or coexpressing WT, +Ala, 3xAla_618-620, or 3xAla_615-617 DAT and EGFP-PICK1. The cells were fixed and incubated with primary MAB369 anti-DAT antibody (1:1000), followed by secondary goat anti-rat Alexa Fluor 568 antibody and visualization by confocal fluorescence microscopy, as described in Materials and Methods. The anti-DAT (red) is shown on the left, the EGFP-PICK1 (green) is in the middle, and the overlay is on the right. Scale bar, 10 μm. All pictures have the same magnification.
    Hek293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36760 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC human embryonic kidney hek 293 cells
    (A and B) 1-CP-U-induced cytotoxicity. A total of 5,000 cells were seeded in triplicate in 96-well plates and then incubated with 1-CP-U for five days. The LS174T, CaSki, MRC-5 and <t>HEK-293</t> cells were subject to treatment with 1-CP-U (1.0 μmol/l). The SKOV3, HeLa, SMMC-7721 and A549 cells were treated with increasing concentrations of 1-CP-U (0.7, 1.0 and 1.4 μmol/l). The cell viability was then analyzed by MTT assay. Data are presented as the mean ± standard deviation of three separate experiments. 1-CP-U, 1-calcium phosphate-uracil.
    Human Embryonic Kidney Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek 293 t cells
    HOTAIRM1 regulates autophagy-associated gene expression by competing with the binding sites of miRNAs. ( a ) HOTAIRM1 transcripts bind to AGO2 directly. AGO2 was assayed by western blotting after acquiring the possible protein complex binding to HOTAIRM1. Proteins bound to the antisense of HOTAIRM1 served as loading controls. ( b ) RNA immunoprecipitation was performed to acquire the RNA that interacted with AGO2 protein. The qRT-PCR product of HOTAIRM1 was tested by agarose gel electrophoresis. ( c ) Luciferase reporter assays analyzing the binding of HOTAIRM1 to miRNAs. NC and miRNAs duplexes were co-transfected with psiCHECH-2 plasmids containing the 59 nt of HOTAIRM1-WT or HOTAIRM1-MUT. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity ( n =3 independent experiments performed in triplicate). ( d ) Luciferase reporter assays analyzing the genes potentially influenced by HOTAIRM1. NC and two HOTAIRM1-siRNAs were co-transfected with psiCHECH-2 plasmids with the 59-nt of miRNAs targeting the wild-type or mutant gene position in HEK-293 T cells. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. Values are derived from  n =3 independent experiments, and data are reported as the mean±S.D. ( e ) Western blotting detecting the proteins expression levels of genes regulated by HOTAIRM1. ULK1, E2F1 and DRAM2 were all downregulated when HOTAIRM1 was knocked down in NB4 cells with or without 1  μ M ATRA treatment; GAPDH was used as a loading control. The densitometric ratio normalized to GAPDH was recorded by quantity one. Values are derived from  n =3 independent experiments, and data are reported as the mean±S.D. (down). ( f–h ) The qRT-PCR testing of DRMA1, LC3B and ULK1, three direct targets of E2F1. Experiments were performed in triplicate and are reported as the mean±S.D. ( i ) The qRT-PCR testing of miRNAs in NB4-Lv-NC and NB4-Lv-shHOTAIRM1 cells after treatment with ATRA (1  μ M, 48 h). Experiments were performed in triplicate and normalized to GAPDH. ( j ) The detection of PML-RARA by western bloting after transient overexpression of miR-20a and miR-106b mimics in NB4 cells. Experiments were performed in triplicate. PML-RARA/GAPDH densitometric ratios were recorded. Differences in  c - i  were considered significant at * P
    Hek 293 T Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene hek293 cells
    Macroscopic properties of expressed and native ClC-1 currents. Voltage dependence of average instantaneous (open symbols) and steady-state (closed symbols) ClC-1 currents recorded from mClC-1–expressing <t>HEK293</t> cells (A; n = 12), mClC-1–expressing skeletal myotubes (B; n = 4), and native FDB fibers obtained from 18–20-d-old WT mice (C; n = 8). Instantaneous currents measured in control noninjected myotubes did not display appreciable ClC-1 currents (upright triangles). (D) Superimposed and normalized instantaneous current–voltage relationships obtained from mClC-1– expressing HEK293 cells (squares), myotubes (inverted triangles), and native WT FDB fibers (circles). (E) Average relative Po - V curves for mClC-1–expressing HEK293 cells (squares), myotubes (inverted triangles), and native WT FDB fibers (circles) obtained from tail currents elicited at −100 mV. Smooth curves through each P O - V dataset were generated using a modified Boltzmann equation ( Eq. 3 ).
    Hek293 Cells, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 781 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Mirus Bio hek293 cells
    Utilization of pMVP for the creation of unique PDX1 vectors. pENTR plasmids containing cDNA for human PDX1 with or without a stop codon (i.e. open) were recombined with pMVP components to generate an assortment of PDX1-expressing ( A ) adenovirus and ( C ) expression plasmid vectors. ( B ) Immunoblot blot analysis of INS1 832/13 cell lysates harvested 48h after treatment with crude adenovirus lysates. For the epitope tagged conditions, note the appearance of the endogenous (lower) and overexpressed (upper) PDX1 bands (top blot). For eGFP blot, the use of P2A adds 23 amino acids to the N-terminal protein (i.e. eGFP). Immunoblot analysis of <t>HEK293</t> cell lysates 24 h after transfection of expression vectors encoding PDX1 with C-terminal ( D ) epitope tags, ( E ) eGFP reporter or fusion, ( G ) and mCherry reporter or fusion. ( F, H ) Fluorescence microscopy of the eGFP and mCherry containing conditions analyzed in panels E and G. Visible bands resulting from ‘Uncleaved’ P2A products (red circle •) and degradation products (*) are labeled.
    Hek293 Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 93/100, based on 1238 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DSMZ hek293 cells
    Larger deletions within the stalk region abrogate ADAM10 and ADAM17 mediated shedding. <t>HEK293</t> cells were transfected with expression plasmids encoding B IL-6RΔA333_V362 ( A ), IL-6RΔE317_T352 ( C and D ), IL-6RΔA323_V362 ( E and F ),
    Hek293 Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 92/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    InvivoGen hek293 cells
    Mean luciferase activity of <t>HEK293</t> TLR reporter cell lines incubated with different fibers (at approx. 400 μg/mL) and positive controls [TNF (5 ng/mL), Pam3-CSK 4 (200 ng/mL), and LPS (250 ng/mL)]. Error bars represent the standard error of the mean values. Asterisks represent classes of statistically significant different responses compared to the medium control (* P
    Hek293 Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 1026 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Corning Life Sciences hek293 cells
    PP2A activity is depressed in vivo by expression of pseudophosphorylated B56α in <t>HEK293</t> cells. A , protein phosphatase ( PP ) activity was determined in reaction mixtures containing either purified heterodimeric PP2A CA or homogenized HEK293 cells
    Hek293 Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson hek293 cells
    Assessment of mitoribosomal misreading in in organello translation Autoradiography of in organello mitochondrial translation products derived from <t>HEK293</t> wild‐type cells in the presence of tobramycin, incorporation of 35 S‐Met and 35 S‐Cys in MT‐CO1 protein. Following in organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in organello translation in the presence of 1,000 μM tobramycin. MT‐CO1 bands were quantified and ratio of 35 S‐Cys/ 35 S‐Met calculated, and the 35 S‐Cys/ 35 S‐Met ratio in the absence of tobramycin was set as 1 ( n = 3), error bars indicate SEM. Ratio of 35 S‐Cys/ 35 S‐Met‐labeled MT‐CO1 and MT‐CO2 proteins synthesized in in organello translation and effect of tobramycin. Following in organello translation in the presence of tobramycin (1,000 μM, 2,000 μM), proteins were immunoprecipitated and analyzed by autoradiography. MT‐CO1 and MT‐CO2 bands were quantified, and ratio of 35 S‐Cys/ 35 S‐Met incorporation was calculated ( n = 3), error bars indicate SEM; * P
    Hek293 Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc hek293 cells
    GP73 interacts and co-localizes with MAVS and TRAF6. ( A ) <t>HEK293</t> cells (1×10 5 ) were co-transfected with GP73 expressing plasmid (0.1 μg) and ISRE or NF-κB reporters (0.05 μg) along with other plasmids as indicated (0.1 μg). Luciferase assays were performed 20 h after transfection. ( B ) HEK293 cells (2×10 6 ) were co-transfected with GP73 expressing plasmid (1 μg) and plasmids as indicated (3 μg) for 24 h. Cells were lysed and lysates were immunoprecipitated with anti-Flag or IgG. Immunoprecipitates and whole cell lysates (WCLs) were analyzed by WB with anti-HA or anti-Flag. ( C ) HEK293 cells (2×10 6 ) were co-transfected with HA- GP73-Myc (2 μg) and Flag- MAVS or Flag- TRAF6 (3 μg) for 24 h. Cells were lysed and lysates were immunoprecipitated with anti-Flag. Immunoprecipitates and WCLs were analyzed by WB with indicated antibodies. ( D ) HEK293 cells (2×10 7 ) were infected with SeV for indicated times. Immunoprecipitation and WB analysis were performed with antibodies against indicated proteins. ( E ) Huh7-MAVSR cells (5×10 7 ) were infected with HCV (MOI = 2) for the indicated days. Immunoprecipitation and WB analysis were performed with antibodies against indicated proteins. ( F ) HEK293 cells (5×10 5 ) were transfected with plasmids as indicated (2 μg) for 24 h. Cell lysates were subjected to GST pull-down assays with equal molar quantity of purified GST (10 μg) or recombinant GST-GP73 (20 μg) proteins. Immunoblots were performed with indicated antibodies. ( G ) HeLa cells (1×10 5 ) were co-transfected with HA-tagged GP73 plasmid (0.3 μg) and Flag-tagged MAVS or TRAF6 plasmids (0.7 μg) for 36 h. Cells were fixed and stained with a rabbit anti-HA monoclonal antibody (MAb) and a mouse anti-Flag MAb, followed by the FITC-conjugated goat anti-rabbit secondary antibody and a DyLight649-conjugated goat anti-mouse secondary antibody before confocal microscopy analysis. Nuclei were counterstained with DAPI. Scale bar, 10 μm. ( H ) HEK293 cells (1×10 7 ) were left un-treated or treated with SeV for 8 h and fractionated with differential centrifugation, the P5K and P50K fractions were subjected to WB with the indicated antibodies. Bar graphs represent means ± SD, ** P
    Hek293 Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1050 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Lysate-based CETSA for SLC1A2 transiently expressed in HEK293 cells. (a) Chemical structure of WAY-213613. (b) The results of immunoblotting show the thermostability of FLAG-SLC1A2 following heating in the presence (+) or absence (−) of WAY-213613 (100 μM). For melting curve analysis, the signal intensity of the thermostable protein was normalized to the intensity of the 45 °C sample. Data are means ± the standard deviation of biological triplicate measurements. The immunoblots are representative results of three biological replicates. Tubulin was analyzed as a control. Legend: L.E., long exposure; WAY, WAY-213613.

    Journal: ACS Chemical Biology

    Article Title: Detection of Chemical Engagement of Solute Carrier Proteins by a Cellular Thermal Shift Assay

    doi: 10.1021/acschembio.8b00270

    Figure Lengend Snippet: Lysate-based CETSA for SLC1A2 transiently expressed in HEK293 cells. (a) Chemical structure of WAY-213613. (b) The results of immunoblotting show the thermostability of FLAG-SLC1A2 following heating in the presence (+) or absence (−) of WAY-213613 (100 μM). For melting curve analysis, the signal intensity of the thermostable protein was normalized to the intensity of the 45 °C sample. Data are means ± the standard deviation of biological triplicate measurements. The immunoblots are representative results of three biological replicates. Tubulin was analyzed as a control. Legend: L.E., long exposure; WAY, WAY-213613.

    Article Snippet: HEK293 cells were obtained from ATCC and grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% (v/v) fetal bovine serum, 100 units mL–1 penicillin, and 100 units mL–1 streptomycin.

    Techniques: Standard Deviation, Western Blot

    CETSA for SLC16A1 inhibitors in HEK293 cell lysates and intact cells. The results of immunoblotting show the thermostability of SLC16A1 and SLCO1A2 following heat treatment at the indicated temperatures in the presence (+) or absence (−) of (a) AZD3965 (50 nM) and (b) AR-C155858 (50 nM). (c) For analysis of dose responses, levels of thermostable SLC16A1 and SLCO1A2 were analyzed in lysates heated to 65 or 75 °C, at 0, 5, 10, 20, 50, 100, and 1000 nM AZD3965. (d) For a cell-based CETSA, cells were incubated with AZD3965 (20 nM). The results of the immunoblot show the increased thermostability of SLC16A1 and SLCO1A2 following the indicated heat treatment. The expression of tubulin was analyzed as a loading control. For quantification of thermostable proteins, the signal intensity was normalized to the respective intensity at 45 °C. In the isothermal dose–response fingerprint (c), the blot intensity was normalized to samples treated with 1000 nM AZD3965. Data are means ± the standard deviation of biological triplicate measurements. The immunoblots are representative of three biological replicates. Legend: AR-C, AR-C155858; L.E., long exposure; Temp., temperature.

    Journal: ACS Chemical Biology

    Article Title: Detection of Chemical Engagement of Solute Carrier Proteins by a Cellular Thermal Shift Assay

    doi: 10.1021/acschembio.8b00270

    Figure Lengend Snippet: CETSA for SLC16A1 inhibitors in HEK293 cell lysates and intact cells. The results of immunoblotting show the thermostability of SLC16A1 and SLCO1A2 following heat treatment at the indicated temperatures in the presence (+) or absence (−) of (a) AZD3965 (50 nM) and (b) AR-C155858 (50 nM). (c) For analysis of dose responses, levels of thermostable SLC16A1 and SLCO1A2 were analyzed in lysates heated to 65 or 75 °C, at 0, 5, 10, 20, 50, 100, and 1000 nM AZD3965. (d) For a cell-based CETSA, cells were incubated with AZD3965 (20 nM). The results of the immunoblot show the increased thermostability of SLC16A1 and SLCO1A2 following the indicated heat treatment. The expression of tubulin was analyzed as a loading control. For quantification of thermostable proteins, the signal intensity was normalized to the respective intensity at 45 °C. In the isothermal dose–response fingerprint (c), the blot intensity was normalized to samples treated with 1000 nM AZD3965. Data are means ± the standard deviation of biological triplicate measurements. The immunoblots are representative of three biological replicates. Legend: AR-C, AR-C155858; L.E., long exposure; Temp., temperature.

    Article Snippet: HEK293 cells were obtained from ATCC and grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% (v/v) fetal bovine serum, 100 units mL–1 penicillin, and 100 units mL–1 streptomycin.

    Techniques: Incubation, Expressing, Standard Deviation, Western Blot

    Conversion of NBCe1 into an anion exchanger. a NBCe1 and AE1 amino-acid alignment showing location of the NBCe1 residues replaced with the corresponding AE1 residues. b Location of the NBCe1 residues replaced with the corresponding AE1 residues located in ion coordination site regions of NBCe1 and AE1 22 (PDB: 4YZF) formed by antiparallel β-strands preceding TMs 3 and 10, and in TM 8 and 10 are shown. c, e-g Loss of Na + -driven base transport and d, h-k gain of Cl − -driven base transport in the mutant constructs. l , m Cell surface expression. Immunoblot analysis of sulfo-NHS-SS-biotin labeled plasma membrane proteins in HEK-293 cells-expressing wt-NBCe1 and NBCe1 mutant (A+B+C). l Cell surface expression was normalized to GAPDH (mutant vs. wt, p

    Journal: Nature Communications

    Article Title: CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1

    doi: 10.1038/s41467-018-03271-3

    Figure Lengend Snippet: Conversion of NBCe1 into an anion exchanger. a NBCe1 and AE1 amino-acid alignment showing location of the NBCe1 residues replaced with the corresponding AE1 residues. b Location of the NBCe1 residues replaced with the corresponding AE1 residues located in ion coordination site regions of NBCe1 and AE1 22 (PDB: 4YZF) formed by antiparallel β-strands preceding TMs 3 and 10, and in TM 8 and 10 are shown. c, e-g Loss of Na + -driven base transport and d, h-k gain of Cl − -driven base transport in the mutant constructs. l , m Cell surface expression. Immunoblot analysis of sulfo-NHS-SS-biotin labeled plasma membrane proteins in HEK-293 cells-expressing wt-NBCe1 and NBCe1 mutant (A+B+C). l Cell surface expression was normalized to GAPDH (mutant vs. wt, p

    Article Snippet: Expression and purification of NBCe1 Human N-terminally Strep(II)-tagged wild-type (wt) NBCe1-A in the pTT vector (Addgene) was transfected into HEK-293 cell (ATCC) monolayers using the calcium phosphate method.

    Techniques: Mutagenesis, Construct, Expressing, Labeling

    Cell viability (EC 50 ) determined by MTT assay for cells incubated with buffer (control); 10 μM phenanthriplatin (Phen); 10 μM cimetidine (Cim); 100 μM cimetidine; 10 μM phenanthriplatin + 10 μM cimetidine; and 10 μM phenanthriplatin + 100 μM cimetidine, for 10 min. (A) HEK 293 WT cells (B) HEK 293 hOCT2 cells (C) HEK293 hMATE1 and hMATE2K cells. Above the columns is the number of experiments. The asterisk ( * ) indicates a statistically significant difference compared to the other columns, # a statistically difference compared to all the other columns except than to 10 μM phenanthriplatin + 10 μM cimetidine (ANOVA).

    Journal: Frontiers in Chemistry

    Article Title: Interaction of the New Monofunctional Anticancer Agent Phenanthriplatin With Transporters for Organic Cations

    doi: 10.3389/fchem.2018.00180

    Figure Lengend Snippet: Cell viability (EC 50 ) determined by MTT assay for cells incubated with buffer (control); 10 μM phenanthriplatin (Phen); 10 μM cimetidine (Cim); 100 μM cimetidine; 10 μM phenanthriplatin + 10 μM cimetidine; and 10 μM phenanthriplatin + 100 μM cimetidine, for 10 min. (A) HEK 293 WT cells (B) HEK 293 hOCT2 cells (C) HEK293 hMATE1 and hMATE2K cells. Above the columns is the number of experiments. The asterisk ( * ) indicates a statistically significant difference compared to the other columns, # a statistically difference compared to all the other columns except than to 10 μM phenanthriplatin + 10 μM cimetidine (ANOVA).

    Article Snippet: Experiments were performed with human embryonic kidney (HEK) 293 cells (CRL-1573; American Type Culture Collection, Rochville, MD), which stably express mOCT1, mOCT2, mOCT3 (Schlatter et al., ), hOCT1, hOCT2, hOCT3, (kind gift of Prof. H. Koepsell, University Würzburg), hMATE1 or hMATE2K (Schmidt-Lauber et al., ).

    Techniques: MTT Assay, Incubation

    Time-lapse experiments on whole HeLa spheroids (A) An anaphase is detected by its peculiar shape at the border of a Hek293 spheroid and the spheroid was continuously imaged. Elapsed times in hours are indicated on the top left of images and the direction of the long axis is denoted by arrows. The bar represents 10 μm. (B) Cells stably expressed survivin-GFP. An area including a metaphase is continuously imaged. Elapsed times are indicated. For each time point, the fluorescent image and the brightfield are shown. A merge and a 2.25-zoom are added on the right; the direction of the fluorescent signal is indicated in red (metaphasic plate from T0 to 28 min and then midbody). The white bar represents 20 μm and the discontinuous one 40 μm. (C) Time-lapse of survivin-GFP in cells grown in 2D. The metaphasic cell enters in anaphase 35 min later and is in telophase at t= 50 min. Based on the fluorescent background a discontinuous line was drawn to figure out the shape of the cell. (D) Same experiment than in B. This larger field included one metaphasic cell and several telophases. The white bar represents 20 μm. Elapsed times are indicated on each photo, T0 been the starting point. (E) Detection of cyclin B on HeLa spheroids. Cyclin B appears in red and DNA detected by hoechst3342 in blue. The arrows point anaphases and a star indicates a metaphase. The bar represented 10 μm.

    Journal: Oncotarget

    Article Title: Unsuccessful mitosis in multicellular tumour spheroids

    doi: 10.18632/oncotarget.15673

    Figure Lengend Snippet: Time-lapse experiments on whole HeLa spheroids (A) An anaphase is detected by its peculiar shape at the border of a Hek293 spheroid and the spheroid was continuously imaged. Elapsed times in hours are indicated on the top left of images and the direction of the long axis is denoted by arrows. The bar represents 10 μm. (B) Cells stably expressed survivin-GFP. An area including a metaphase is continuously imaged. Elapsed times are indicated. For each time point, the fluorescent image and the brightfield are shown. A merge and a 2.25-zoom are added on the right; the direction of the fluorescent signal is indicated in red (metaphasic plate from T0 to 28 min and then midbody). The white bar represents 20 μm and the discontinuous one 40 μm. (C) Time-lapse of survivin-GFP in cells grown in 2D. The metaphasic cell enters in anaphase 35 min later and is in telophase at t= 50 min. Based on the fluorescent background a discontinuous line was drawn to figure out the shape of the cell. (D) Same experiment than in B. This larger field included one metaphasic cell and several telophases. The white bar represents 20 μm. Elapsed times are indicated on each photo, T0 been the starting point. (E) Detection of cyclin B on HeLa spheroids. Cyclin B appears in red and DNA detected by hoechst3342 in blue. The arrows point anaphases and a star indicates a metaphase. The bar represented 10 μm.

    Article Snippet: Cells HeLa and Hek293 cell lines obtained from ATCC were cultured under the suggested conditions.

    Techniques: Stable Transfection

    Time-lapse experiments on whole Hek293 spheroids (A) Hek293 cells expressing histone H2A-GFP are continuously imaged. An early anaphase is selected at T0. The fluorescent signal of histone H2A-GFP, the brightfield and the merge are shown at representative times. (B) A z-stack imaging of the field shown in (A) was performed at T68. The maximum intensity projection of the whole fluorescent signal is represented with a depth colour code. Arrows indicates the two nuclei derived from the anaphase imaged in A (one brown, one green). (C) Same time-lapse as in A performed on whole spheroids kept for one day in Labteck wells. The border cells start to spread on the glass as seen in (a) and the metaphase cells escaping from the spheroid mass undergo normal in mitosis.

    Journal: Oncotarget

    Article Title: Unsuccessful mitosis in multicellular tumour spheroids

    doi: 10.18632/oncotarget.15673

    Figure Lengend Snippet: Time-lapse experiments on whole Hek293 spheroids (A) Hek293 cells expressing histone H2A-GFP are continuously imaged. An early anaphase is selected at T0. The fluorescent signal of histone H2A-GFP, the brightfield and the merge are shown at representative times. (B) A z-stack imaging of the field shown in (A) was performed at T68. The maximum intensity projection of the whole fluorescent signal is represented with a depth colour code. Arrows indicates the two nuclei derived from the anaphase imaged in A (one brown, one green). (C) Same time-lapse as in A performed on whole spheroids kept for one day in Labteck wells. The border cells start to spread on the glass as seen in (a) and the metaphase cells escaping from the spheroid mass undergo normal in mitosis.

    Article Snippet: Cells HeLa and Hek293 cell lines obtained from ATCC were cultured under the suggested conditions.

    Techniques: Expressing, Imaging, Derivative Assay

    Cytokinesis failure and presence of binucleated cells in spheroids (A) In (a) TSA/pc spheroids were grown for 7 days in U-low binding plates; In (b) TSA/pc spheroids prepared by the pending drop technic and recovered for staining on day 7. In (c) Hek293 spheroids were grown, for 7 days, in U-low binding plates. The outlines of cells are highlighted by labelling actin with rhodamin-phalloidin (a, b and c). DNA was labelled by either NucRed in (a) or hoechst 33342 in (b) and (c). Mitotic cells are indicated by an arrow and binucleated cells by stars. The bar represents 20 μm. (B) Estimation of the percentage of binucleated cells in three cell lines. 3 200 and 1 650 TSA/pc, 750 and 640 Hek293 and finally, 889 and 1 544 HeLa cells were scored on 2D-cultures and in D7-spheroids, respectively. Results obtained with 2D-cell cultures are represented by white histograms whereas 3D-spheroids data are in blue. The differences between 2D and 3D were found significant (p

    Journal: Oncotarget

    Article Title: Unsuccessful mitosis in multicellular tumour spheroids

    doi: 10.18632/oncotarget.15673

    Figure Lengend Snippet: Cytokinesis failure and presence of binucleated cells in spheroids (A) In (a) TSA/pc spheroids were grown for 7 days in U-low binding plates; In (b) TSA/pc spheroids prepared by the pending drop technic and recovered for staining on day 7. In (c) Hek293 spheroids were grown, for 7 days, in U-low binding plates. The outlines of cells are highlighted by labelling actin with rhodamin-phalloidin (a, b and c). DNA was labelled by either NucRed in (a) or hoechst 33342 in (b) and (c). Mitotic cells are indicated by an arrow and binucleated cells by stars. The bar represents 20 μm. (B) Estimation of the percentage of binucleated cells in three cell lines. 3 200 and 1 650 TSA/pc, 750 and 640 Hek293 and finally, 889 and 1 544 HeLa cells were scored on 2D-cultures and in D7-spheroids, respectively. Results obtained with 2D-cell cultures are represented by white histograms whereas 3D-spheroids data are in blue. The differences between 2D and 3D were found significant (p

    Article Snippet: Cells HeLa and Hek293 cell lines obtained from ATCC were cultured under the suggested conditions.

    Techniques: Binding Assay, Staining

    Directed evolution of an eFRET GEVI A) A model of QuasAr-mOrange2 constructs, represented by the crystal structures of Arch-2 (PDB ID 2EI4) 26 and mOrange (PDB ID 2H5O) 27 . The color scale represents the degree of order in the crystal structure, as reported by the B-factor. Less ordered regions are presumed more dispensable. B) Design of linker truncation libraries. QuasAr1.2, the best available variant at the time of the screen, was used as the electrochromic quencher. C) Hierarchical screen of truncated linker libraries for eFRET GEVIs. Constructs were first screened in E. coli for mOrange2 brightness and then screened for membrane trafficking in HEK cells. Voltage-sensitivity was then tested via field stimulation in HeLa cells co-expressing the eFRET GEVI, ArcLight (as an internal control), and Kir2.1 (to lower the resting voltage to −60 mV 42 ). D) ArcLight fluorescence of three HeLa cells. Yellow arrow indicates direction of the applied electric field. Scale bar: 25 μm. E) Fluorescence intensity traces of the three regions shown in D). F) mOrange2 fluorescence from the same cells shown in D). G) Response of mOrange2 fluorescence during stimulation as in E). Fluorescence traces show single-trial measurement of cells imaged at 100 frames per second. H) Effect of linker length on voltage sensitivity in QuasAr1.2-mOrange2. For each linker length the most sensitive eFRET GEVI construct was compared to ArcLight measured in the same cells. Error bars represent standard deviation over n = 15–20 cells in the ratio ΔF/F.

    Journal: Nature communications

    Article Title: Bright and fast multi-colored voltage reporters via electrochromic FRET

    doi: 10.1038/ncomms5625

    Figure Lengend Snippet: Directed evolution of an eFRET GEVI A) A model of QuasAr-mOrange2 constructs, represented by the crystal structures of Arch-2 (PDB ID 2EI4) 26 and mOrange (PDB ID 2H5O) 27 . The color scale represents the degree of order in the crystal structure, as reported by the B-factor. Less ordered regions are presumed more dispensable. B) Design of linker truncation libraries. QuasAr1.2, the best available variant at the time of the screen, was used as the electrochromic quencher. C) Hierarchical screen of truncated linker libraries for eFRET GEVIs. Constructs were first screened in E. coli for mOrange2 brightness and then screened for membrane trafficking in HEK cells. Voltage-sensitivity was then tested via field stimulation in HeLa cells co-expressing the eFRET GEVI, ArcLight (as an internal control), and Kir2.1 (to lower the resting voltage to −60 mV 42 ). D) ArcLight fluorescence of three HeLa cells. Yellow arrow indicates direction of the applied electric field. Scale bar: 25 μm. E) Fluorescence intensity traces of the three regions shown in D). F) mOrange2 fluorescence from the same cells shown in D). G) Response of mOrange2 fluorescence during stimulation as in E). Fluorescence traces show single-trial measurement of cells imaged at 100 frames per second. H) Effect of linker length on voltage sensitivity in QuasAr1.2-mOrange2. For each linker length the most sensitive eFRET GEVI construct was compared to ArcLight measured in the same cells. Error bars represent standard deviation over n = 15–20 cells in the ratio ΔF/F.

    Article Snippet: For each variant, the plasma membrane trafficking was examined in HEK cells (CRL-1573™, ATCC) co-expressing ArcLight, which served as internal reference.

    Techniques: Construct, Variant Assay, Expressing, Fluorescence, Standard Deviation

    Voltage sensing with GEVIs in HEK293 cells Voltage response of eFRET GEVI reporters are shown in (A–C), and voltage responses of comparison GEVIs are shown in (D–F). A) and D) Fluorescence images of HEK cells expressing GEVI reporters. Scale bars 10 μm. B) and E) Fluorescence as a function of membrane voltage, normalized to fluorescence at V = −70 mV. Many eFRET-based GEVIs showed a small amount of hysteresis. Each trace is a single trial with 100 ms exposures. C) and F) Fluorescence response to a step in membrane voltage from −70 mV to +30 mV. Each trace is averaged over 20–30 trials, with 1 ms exposures. All data was acquired with laser illumination (3 W cm −2 for eFRET, ASAP1, and ArcLight GEVIs, 200 W cm −2 for QuasAr2). No temporal filtering was applied. Fluorescence traces were corrected for photobleaching. G) Quantification of eFRET GEVI sensitivity and speed. Voltage sensitivity of eFRET GEVIs approximately corresponds to the degree of spectral overlap between the emission of the GEVI and the absorption of QuasAr2. H) eFRET GEVI sensitivity and speed in the context of other GEVIs. Response speed was defined as the inverse of the time to reach 50% of steady-state response to a voltage step from −70 mV to +30 mV. Data in G) and H) represents an average of n = 6 to 9 cells for each construct. Error bars represent s.e.m.

    Journal: Nature communications

    Article Title: Bright and fast multi-colored voltage reporters via electrochromic FRET

    doi: 10.1038/ncomms5625

    Figure Lengend Snippet: Voltage sensing with GEVIs in HEK293 cells Voltage response of eFRET GEVI reporters are shown in (A–C), and voltage responses of comparison GEVIs are shown in (D–F). A) and D) Fluorescence images of HEK cells expressing GEVI reporters. Scale bars 10 μm. B) and E) Fluorescence as a function of membrane voltage, normalized to fluorescence at V = −70 mV. Many eFRET-based GEVIs showed a small amount of hysteresis. Each trace is a single trial with 100 ms exposures. C) and F) Fluorescence response to a step in membrane voltage from −70 mV to +30 mV. Each trace is averaged over 20–30 trials, with 1 ms exposures. All data was acquired with laser illumination (3 W cm −2 for eFRET, ASAP1, and ArcLight GEVIs, 200 W cm −2 for QuasAr2). No temporal filtering was applied. Fluorescence traces were corrected for photobleaching. G) Quantification of eFRET GEVI sensitivity and speed. Voltage sensitivity of eFRET GEVIs approximately corresponds to the degree of spectral overlap between the emission of the GEVI and the absorption of QuasAr2. H) eFRET GEVI sensitivity and speed in the context of other GEVIs. Response speed was defined as the inverse of the time to reach 50% of steady-state response to a voltage step from −70 mV to +30 mV. Data in G) and H) represents an average of n = 6 to 9 cells for each construct. Error bars represent s.e.m.

    Article Snippet: For each variant, the plasma membrane trafficking was examined in HEK cells (CRL-1573™, ATCC) co-expressing ArcLight, which served as internal reference.

    Techniques: Fluorescence, Expressing, Mass Spectrometry, Construct

    sFRP3 with the Arg324Gly substitution is less efficient at antagonizing wnt signaling. ( A ) HEK293 cells were transfected with 1.0 μg, 0.5 μg, 0.25 μg, or 0.125 μg of wild-type (wt) pcFRZB, pcFRZB-Arg200Trp, pcFRZB-Arg324Gly, or pcFRZB Arg200Trp/Arg324Gly or were left untransfected (Control, C ). After 24 h, the cells were lysed and 30 μg of protein was separated by SDS/PAGE under reducing conditions. The protein was transferred to a polyvinylidine difloride membrane and then serially probed with anti-sFRP3 and β-actin. ( B ) HEK293 cells were cotransfected in triplicate with 0.25 μg of pTOPFLASH-Luc, 0.025 μg of pACB-Z, 0.25 μg of pUSE-Wnt1, and 1 μg of either pcFRZB, pcFRZB-Arg200Trp, pcFRZB-Arg324Gly, pcFRZB-Arg200Trp/Arg324Gly, or vector DNA. ( C ) Similar transfections were performed with 0.25 μg of a β-catenin-expressing plasmid instead of the Wnt1-expressing plasmid. Control cultures were transfected with pTOPFLASH-Luc and pACB-Z to establish basal enzyme activity levels. Additional control cultures were transfected with pTOPFLASH-Luc, which confirmed specificity of the assay (data not shown). After 24 h, the cells were lysed and the normalized luciferase activities were determined. The fold induction values are the ratios of the normalized luciferase activities in cells transfected with both expression and reporter plasmids compared with activities in cells receiving the respective reporter plasmids alone. Shown are the mean ± SEM of triplicates. ( D ) Triplicate cultures of HEK293 cells were transfected with 1 μg, 0.3 μg, or 0.1 μg of pcFRZB or pcFRZB-Arg324Gly. Control cultures were transfected with empty vector. After 48 h, the cells were lysed and the cytosolic and nuclear fractions were separated by SDS/PAGE, transferred to a polyvinylidine difloride membrane, and then probed with antibodies for β-catenin and β-actin.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females

    doi: 10.1073/pnas.0403456101

    Figure Lengend Snippet: sFRP3 with the Arg324Gly substitution is less efficient at antagonizing wnt signaling. ( A ) HEK293 cells were transfected with 1.0 μg, 0.5 μg, 0.25 μg, or 0.125 μg of wild-type (wt) pcFRZB, pcFRZB-Arg200Trp, pcFRZB-Arg324Gly, or pcFRZB Arg200Trp/Arg324Gly or were left untransfected (Control, C ). After 24 h, the cells were lysed and 30 μg of protein was separated by SDS/PAGE under reducing conditions. The protein was transferred to a polyvinylidine difloride membrane and then serially probed with anti-sFRP3 and β-actin. ( B ) HEK293 cells were cotransfected in triplicate with 0.25 μg of pTOPFLASH-Luc, 0.025 μg of pACB-Z, 0.25 μg of pUSE-Wnt1, and 1 μg of either pcFRZB, pcFRZB-Arg200Trp, pcFRZB-Arg324Gly, pcFRZB-Arg200Trp/Arg324Gly, or vector DNA. ( C ) Similar transfections were performed with 0.25 μg of a β-catenin-expressing plasmid instead of the Wnt1-expressing plasmid. Control cultures were transfected with pTOPFLASH-Luc and pACB-Z to establish basal enzyme activity levels. Additional control cultures were transfected with pTOPFLASH-Luc, which confirmed specificity of the assay (data not shown). After 24 h, the cells were lysed and the normalized luciferase activities were determined. The fold induction values are the ratios of the normalized luciferase activities in cells transfected with both expression and reporter plasmids compared with activities in cells receiving the respective reporter plasmids alone. Shown are the mean ± SEM of triplicates. ( D ) Triplicate cultures of HEK293 cells were transfected with 1 μg, 0.3 μg, or 0.1 μg of pcFRZB or pcFRZB-Arg324Gly. Control cultures were transfected with empty vector. After 48 h, the cells were lysed and the cytosolic and nuclear fractions were separated by SDS/PAGE, transferred to a polyvinylidine difloride membrane, and then probed with antibodies for β-catenin and β-actin.

    Article Snippet: The human embryonic kidney cell line HEK293 (American Type Culture Collection) was transfected by using SuperFect (Qiagen, Valencia, CA).

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

    Evaluation of GB toxicity on HEK-293 cell viability. The cells were grown in a range of GB concentrations (0.125–2.000 mg/mL) for 24, 48, or 72 h. Cell viability was measured using the MTT test. ** ( p

    Journal: Molecules

    Article Title: Antioxidant Sulfated Polysaccharide from Edible Red Seaweed Gracilaria birdiae is an Inhibitor of Calcium Oxalate Crystal Formation

    doi: 10.3390/molecules25092055

    Figure Lengend Snippet: Evaluation of GB toxicity on HEK-293 cell viability. The cells were grown in a range of GB concentrations (0.125–2.000 mg/mL) for 24, 48, or 72 h. Cell viability was measured using the MTT test. ** ( p

    Article Snippet: The HEK-293 cells (ATCC® CRL-1573™) were grown in a DMEM medium with 10% FBS, 100 μg/mL streptomycin and 100 IU/mL penicillin.

    Techniques: MTT Assay

    DN-Pin1 inhibits the EGF induced p-NF-H in transfected HEK293 cells. A , Western blot analysis of NF-H phosphorylation. HEK293 cells were transfected with NF-H ( lanes 1-4 ) or cotransfected with DN-Pin1 ( lane 4 ). After transfection for 24 h, cells were

    Journal:

    Article Title: Pin1-dependent Prolyl Isomerization Modulates the Stress-induced Phosphorylation of High Molecular Weight Neurofilament Protein *

    doi: 10.1074/jbc.M801633200

    Figure Lengend Snippet: DN-Pin1 inhibits the EGF induced p-NF-H in transfected HEK293 cells. A , Western blot analysis of NF-H phosphorylation. HEK293 cells were transfected with NF-H ( lanes 1-4 ) or cotransfected with DN-Pin1 ( lane 4 ). After transfection for 24 h, cells were

    Article Snippet: HEK293 Cell Culture and Transfection —HEK293 cells were obtained from the American Type Culture Collection, cultured in Dulbecco's modified Eagle's medium with 10% calf serum, and supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques: Transfection, Western Blot

    Mfsd2a KO mice have decreased liver TG with normal liver metabolism. Oil red-O histology of fasted A, WT and B, KO liver illustrates neutral lipid stores. C, Quantification of TG extracted from fed and fasted WT and KO livers. D, Assay of β-oxidation in liver homogenates from fasted WT and KO littermates (n = 3–4 per genotype, data representative of three independent experiments). E, In vivo uptake of 14 C-oleate in indicated tissues from WT and KO mice, normalized to heart uptake (n = 3 per genotype). F, TLC of lipid extract illustrating in vitro incorporation of 14 C-glycerol and 14 C-oleate by control and Mfsd2a-transfected HEK293 cells (n = 3). CE, cholesterol ester; M/D/TAG, mono-, di-, tri-acylglycerol; PL, phospholipids. G-H, In vivo VLDL production was assayed by measuring G, serum TG and H, cholesterol in fasted WT and KO mice (n > 5 per genotype) following treatment with LPL-inhibitor F-127. I, Real-time PCR analysis of PPARα targets and fatty acid synthesis genes from fasted WT and KO liver (n = 3–4). J , Pyruvate tolerance test of 12–14-week old chow-fed WT and KO littermates after an overnight fast (n = 6). Data are displayed as mean ± SEM. * p

    Journal: PLoS ONE

    Article Title: Major Facilitator Superfamily Domain-Containing Protein 2a (MFSD2A) Has Roles in Body Growth, Motor Function, and Lipid Metabolism

    doi: 10.1371/journal.pone.0050629

    Figure Lengend Snippet: Mfsd2a KO mice have decreased liver TG with normal liver metabolism. Oil red-O histology of fasted A, WT and B, KO liver illustrates neutral lipid stores. C, Quantification of TG extracted from fed and fasted WT and KO livers. D, Assay of β-oxidation in liver homogenates from fasted WT and KO littermates (n = 3–4 per genotype, data representative of three independent experiments). E, In vivo uptake of 14 C-oleate in indicated tissues from WT and KO mice, normalized to heart uptake (n = 3 per genotype). F, TLC of lipid extract illustrating in vitro incorporation of 14 C-glycerol and 14 C-oleate by control and Mfsd2a-transfected HEK293 cells (n = 3). CE, cholesterol ester; M/D/TAG, mono-, di-, tri-acylglycerol; PL, phospholipids. G-H, In vivo VLDL production was assayed by measuring G, serum TG and H, cholesterol in fasted WT and KO mice (n > 5 per genotype) following treatment with LPL-inhibitor F-127. I, Real-time PCR analysis of PPARα targets and fatty acid synthesis genes from fasted WT and KO liver (n = 3–4). J , Pyruvate tolerance test of 12–14-week old chow-fed WT and KO littermates after an overnight fast (n = 6). Data are displayed as mean ± SEM. * p

    Article Snippet: Cell Culture HEK293 cells (ATCC) were cultured in DMEM (Invitrogen) with 10% fetal bovine serum and penicillin/streptomycin at 37°C with 5% CO2 .

    Techniques: Mouse Assay, In Vivo, Thin Layer Chromatography, In Vitro, Transfection, Real-time Polymerase Chain Reaction

    MFSD2A is rapidly turned over from the cell surface via the lysosome. A, MFSD2A turnover as a function of time in cycloheximide-treated (CHX) HEK293 cells transfected with mouse Mfsd2a and primary hepatocytes from fasted mice. Time indicates duration of CHX treatment. Graph quantifies MFSD2A expression. B, Turnover of plasma membrane MFSD2A in Mfsd2a-transfected HEK293 cells. Cells were incubated with CHX for the indicated time period and then treated with membrane-impermeable maleimide-PEG 2 -Biotin. Protein homogenates were immunoprecipitated with streptavidin beads, prior to Western blot analysis for plasma membrane localized MFSD2A. C and D, Mfsd2a-transfected HEK293 cells were treated with CHX as in panel A , with or without ( C ) chloroquine (CLQ) or ( D ) MG-132 (Western blot not shown). Protein extracts were analyzed by Western blot for MFSD2A. Graphs quantify MFSD2A expression. β-actin in A and C and Na-K-ATPase in B served as a loading control. Quantification represents experiments in triplicate, displayed as mean ± SD.

    Journal: PLoS ONE

    Article Title: Major Facilitator Superfamily Domain-Containing Protein 2a (MFSD2A) Has Roles in Body Growth, Motor Function, and Lipid Metabolism

    doi: 10.1371/journal.pone.0050629

    Figure Lengend Snippet: MFSD2A is rapidly turned over from the cell surface via the lysosome. A, MFSD2A turnover as a function of time in cycloheximide-treated (CHX) HEK293 cells transfected with mouse Mfsd2a and primary hepatocytes from fasted mice. Time indicates duration of CHX treatment. Graph quantifies MFSD2A expression. B, Turnover of plasma membrane MFSD2A in Mfsd2a-transfected HEK293 cells. Cells were incubated with CHX for the indicated time period and then treated with membrane-impermeable maleimide-PEG 2 -Biotin. Protein homogenates were immunoprecipitated with streptavidin beads, prior to Western blot analysis for plasma membrane localized MFSD2A. C and D, Mfsd2a-transfected HEK293 cells were treated with CHX as in panel A , with or without ( C ) chloroquine (CLQ) or ( D ) MG-132 (Western blot not shown). Protein extracts were analyzed by Western blot for MFSD2A. Graphs quantify MFSD2A expression. β-actin in A and C and Na-K-ATPase in B served as a loading control. Quantification represents experiments in triplicate, displayed as mean ± SD.

    Article Snippet: Cell Culture HEK293 cells (ATCC) were cultured in DMEM (Invitrogen) with 10% fetal bovine serum and penicillin/streptomycin at 37°C with 5% CO2 .

    Techniques: Transfection, Mouse Assay, Expressing, Incubation, Immunoprecipitation, Western Blot

    Disease-associated mutations increase temperature activation of TRPM3. HEK293 cells were transfected with the human TRPM3α2, or its mutants; fura-2 Ca 2+ imaging experiments were performed as described in the Materials and methods section. ( A-C ) Averaged fluorescence ratio traces (340 nm/380 nm) for all PregS-responsive cells from one coverslip in cells expressing TRPM3 ( A ), V992M ( B ) and P1092Q ( C ). The cells were first stimulated by increasing the temperature to 37°C, followed by 10 μM primidone at room temperature to facilitate return of Ca 2+ to baseline, finally 25 μM PregS was applied. ( D ) The change in 340/380 ratio induced by 37°C expressed as a fraction of the response to 25 μM PregS. ( E ) 340/380 ratios at baseline, in response to 37°C and in response to 25 μM PregS. Each symbol represents the average value from all PregS-responsive cells from one coverslip, lines connect data points from the same coverslip. Each coverslips had around 70–80 PregS-responsive cells in the WT group; 35–45 for P1092Q, and 15–25 for V992M. Data were collected from three independent transfections.

    Journal: eLife

    Article Title: Disease-associated mutations in the human TRPM3 render the channel overactive via two distinct mechanisms

    doi: 10.7554/eLife.55634

    Figure Lengend Snippet: Disease-associated mutations increase temperature activation of TRPM3. HEK293 cells were transfected with the human TRPM3α2, or its mutants; fura-2 Ca 2+ imaging experiments were performed as described in the Materials and methods section. ( A-C ) Averaged fluorescence ratio traces (340 nm/380 nm) for all PregS-responsive cells from one coverslip in cells expressing TRPM3 ( A ), V992M ( B ) and P1092Q ( C ). The cells were first stimulated by increasing the temperature to 37°C, followed by 10 μM primidone at room temperature to facilitate return of Ca 2+ to baseline, finally 25 μM PregS was applied. ( D ) The change in 340/380 ratio induced by 37°C expressed as a fraction of the response to 25 μM PregS. ( E ) 340/380 ratios at baseline, in response to 37°C and in response to 25 μM PregS. Each symbol represents the average value from all PregS-responsive cells from one coverslip, lines connect data points from the same coverslip. Each coverslips had around 70–80 PregS-responsive cells in the WT group; 35–45 for P1092Q, and 15–25 for V992M. Data were collected from three independent transfections.

    Article Snippet: HEK293 cells were cultured in MEM supplemented with 10% FBS and 100 IU/ml penicillin plus 100 µg/ml streptomycin in 5% CO2 at 37°C.

    Techniques: Activation Assay, Transfection, Imaging, Fluorescence, Expressing

    PregS responses of wild type and mutant TRPM3 at 37°C. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) as described in the Materials and method section. ( A ) Average traces from one plate (four wells for each condition) for stimulation with different concentrations of PregS for cells expressing wild type TRPM3; at the end of the experiment, 2 μM ionomycin was applied as a normalizing stimulus. ( B ) Hill fit of the PregS concentration response relationship, symbols show values obtained in individual wells n = 8 from two independent transfections. The dashed line shows the fit for the concentration response curve obtained at 21°C from Figure 1D . ( C-D ) Identical experiments to those shown in panel A, on cells expressing the V992M (C) and the P1092Q (D) mutants.

    Journal: eLife

    Article Title: Disease-associated mutations in the human TRPM3 render the channel overactive via two distinct mechanisms

    doi: 10.7554/eLife.55634

    Figure Lengend Snippet: PregS responses of wild type and mutant TRPM3 at 37°C. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) as described in the Materials and method section. ( A ) Average traces from one plate (four wells for each condition) for stimulation with different concentrations of PregS for cells expressing wild type TRPM3; at the end of the experiment, 2 μM ionomycin was applied as a normalizing stimulus. ( B ) Hill fit of the PregS concentration response relationship, symbols show values obtained in individual wells n = 8 from two independent transfections. The dashed line shows the fit for the concentration response curve obtained at 21°C from Figure 1D . ( C-D ) Identical experiments to those shown in panel A, on cells expressing the V992M (C) and the P1092Q (D) mutants.

    Article Snippet: HEK293 cells were cultured in MEM supplemented with 10% FBS and 100 IU/ml penicillin plus 100 µg/ml streptomycin in 5% CO2 at 37°C.

    Techniques: Mutagenesis, Transfection, Fluorescence, Expressing, Concentration Assay

    Disease-associate mutations increase temperature sensitivity of TRPM3. HEK293 cells were transfected with the human TRPM3α2, or its mutants; whole-cell patch clamp electrophysiology was performed as descried in the Materials and methods section using a ramp protocol from −100 to 100 mV. ( A-C ) Representative measurements, top panels show temperature recordings, bottom panels show currents at 100 mV and −100 mV. The applications of 100 μM PregS are indicated by the horizontal lines. ( D-F ) The heat-induced current amplitudes at 100 mV were normalized to the currents induced by PregS and plotted as a function of the temperature from the same data presented in panels A-C. ( G ) Summary of the slopes of the current increases between 23°C and 33°C determined from linear fits from panels D-F. ( H ) Summary of current amplitudes at 100 and −100 mV induced by increasing the temperature to 25°C, 30°C and 35°C as well as in response to PregS.

    Journal: eLife

    Article Title: Disease-associated mutations in the human TRPM3 render the channel overactive via two distinct mechanisms

    doi: 10.7554/eLife.55634

    Figure Lengend Snippet: Disease-associate mutations increase temperature sensitivity of TRPM3. HEK293 cells were transfected with the human TRPM3α2, or its mutants; whole-cell patch clamp electrophysiology was performed as descried in the Materials and methods section using a ramp protocol from −100 to 100 mV. ( A-C ) Representative measurements, top panels show temperature recordings, bottom panels show currents at 100 mV and −100 mV. The applications of 100 μM PregS are indicated by the horizontal lines. ( D-F ) The heat-induced current amplitudes at 100 mV were normalized to the currents induced by PregS and plotted as a function of the temperature from the same data presented in panels A-C. ( G ) Summary of the slopes of the current increases between 23°C and 33°C determined from linear fits from panels D-F. ( H ) Summary of current amplitudes at 100 and −100 mV induced by increasing the temperature to 25°C, 30°C and 35°C as well as in response to PregS.

    Article Snippet: HEK293 cells were cultured in MEM supplemented with 10% FBS and 100 IU/ml penicillin plus 100 µg/ml streptomycin in 5% CO2 at 37°C.

    Techniques: Transfection, Patch Clamp

    Primidone inhibits Ca 2+ responses induced by EC 50 concentrations of PregS. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) at room temperature (~21°C) as described in the Materials and method section. ( A-C ) Cells were stimulated by PregS at its EC 50 concentration for each construct, as indicated by the first arrow, then various concentrations of primidone were applied (second arrow). ( D ) Hill1 fits of the concentration dependence of primidone; symbols show mean ± SEM from five or six wells, from two independent transfections. The IC 50 values were 2.37 ± 0.39 μM for wild-type channels, 8.62 ± 1.42 μM for V992M and 3.52 ± 1.13 μM for P1092Q.

    Journal: eLife

    Article Title: Disease-associated mutations in the human TRPM3 render the channel overactive via two distinct mechanisms

    doi: 10.7554/eLife.55634

    Figure Lengend Snippet: Primidone inhibits Ca 2+ responses induced by EC 50 concentrations of PregS. HEK293 cells were transfected with the Ca 2+ indicator GCaMP6f and the hTRPM3α2 or its mutants, and fluorescence was measured in a 96-well plate reader (Flexstation-3) at room temperature (~21°C) as described in the Materials and method section. ( A-C ) Cells were stimulated by PregS at its EC 50 concentration for each construct, as indicated by the first arrow, then various concentrations of primidone were applied (second arrow). ( D ) Hill1 fits of the concentration dependence of primidone; symbols show mean ± SEM from five or six wells, from two independent transfections. The IC 50 values were 2.37 ± 0.39 μM for wild-type channels, 8.62 ± 1.42 μM for V992M and 3.52 ± 1.13 μM for P1092Q.

    Article Snippet: HEK293 cells were cultured in MEM supplemented with 10% FBS and 100 IU/ml penicillin plus 100 µg/ml streptomycin in 5% CO2 at 37°C.

    Techniques: Transfection, Fluorescence, Concentration Assay, Construct

    Detection of selectin receptors with antibodies and fusion proteins on HEK-293 EBOV-GP cells (6a) Flow cytometry analysis showing the binding of different monoclonal antibodies against CD44v6, CD44s, DR3, CD24, HLAabc, and NCAM (CD56) to HEK-293 EBOV-GP cells compared to vector transfected controls. (6b), Binding of NKG2D-Fc to its ligands MICA/B (MHC I chain related molecule) is reduced in HEK-293 transfected cells either with the Marburg-GP or the 7916-5 GP, while binding of CD24 and CD44s is significantly increased. The expression of HLA is reduced. The binding of mAb against CD44v6, DR3 is not significantly changed.

    Journal: bioRxiv

    Article Title: Activating NK- receptors, homing selectins and inhibitory Siglecs recognize EBOLA-GP and HPV-L1NK

    doi: 10.1101/2020.07.24.219329

    Figure Lengend Snippet: Detection of selectin receptors with antibodies and fusion proteins on HEK-293 EBOV-GP cells (6a) Flow cytometry analysis showing the binding of different monoclonal antibodies against CD44v6, CD44s, DR3, CD24, HLAabc, and NCAM (CD56) to HEK-293 EBOV-GP cells compared to vector transfected controls. (6b), Binding of NKG2D-Fc to its ligands MICA/B (MHC I chain related molecule) is reduced in HEK-293 transfected cells either with the Marburg-GP or the 7916-5 GP, while binding of CD24 and CD44s is significantly increased. The expression of HLA is reduced. The binding of mAb against CD44v6, DR3 is not significantly changed.

    Article Snippet: P24 antigen capture assay Lentivirus particles based on the human immunodeficiency virus-1 (HIV-1) were produced in HEK-293 cells through transient transfection of 2 plasmids encoding components of the virus envelope.

    Techniques: Flow Cytometry, Binding Assay, Plasmid Preparation, Transfection, Expressing

    Lysis-reduction of HEK-293 EBOV-GP target cells by polyclonal NK-cells. (10a) NK activation receptors NKp44, NKp46, inhibitory Siglecs and L-selectin are able to bind strongly to HEK-293 EBOV-GP cells, compared to mock transfected HEK-293 cells. The results of a chromium release assay indicate that HEK-293 target cells show decreased susceptibility to lysis by polyclonal NK-cells. In this assay, specific anti-NCRs antibodies blocked the interaction between NCRs and their ligands on HEK-293 cells. (10b) Results of a chromium release assay using HEK-293 target cells transfected with EBOV-GP show decreased susceptibility to lysis by polyclonal NK-cells in comparison to non-transfected HEK-293 cells.

    Journal: bioRxiv

    Article Title: Activating NK- receptors, homing selectins and inhibitory Siglecs recognize EBOLA-GP and HPV-L1NK

    doi: 10.1101/2020.07.24.219329

    Figure Lengend Snippet: Lysis-reduction of HEK-293 EBOV-GP target cells by polyclonal NK-cells. (10a) NK activation receptors NKp44, NKp46, inhibitory Siglecs and L-selectin are able to bind strongly to HEK-293 EBOV-GP cells, compared to mock transfected HEK-293 cells. The results of a chromium release assay indicate that HEK-293 target cells show decreased susceptibility to lysis by polyclonal NK-cells. In this assay, specific anti-NCRs antibodies blocked the interaction between NCRs and their ligands on HEK-293 cells. (10b) Results of a chromium release assay using HEK-293 target cells transfected with EBOV-GP show decreased susceptibility to lysis by polyclonal NK-cells in comparison to non-transfected HEK-293 cells.

    Article Snippet: P24 antigen capture assay Lentivirus particles based on the human immunodeficiency virus-1 (HIV-1) were produced in HEK-293 cells through transient transfection of 2 plasmids encoding components of the virus envelope.

    Techniques: Lysis, Activation Assay, Transfection, Release Assay

    Expression efficiency of EBOV-GP in transfected CHOK1- and HEK-293 cells. Plasmids from Marburg (pCAGGS.cm5 + GP) and Heidelberg (pcDNA™6/V5-His + GP) were used for transfection of HEK-293 cells (3a), while the the plasmid pCDNA 3.1+ GP was used in CHO-K1 cells (3b). GP expression was detected by anti-ZEBOV GP mouse antibody (3B11), and anti-ZEBOV GP human antibody (KZ52). The binding to EBOV-GP transfected HEK-293-cells by the anti-GP mouse antibody 3B11 was trypsin-dependent (3a left). The human anti-GP did not show a dependency on trypsin (3a right). After transfection, the strongly positive cells were sorted and further checked for their degree of EBOV-GP expression by mAb anti EBOV-GP (KZ52).

    Journal: bioRxiv

    Article Title: Activating NK- receptors, homing selectins and inhibitory Siglecs recognize EBOLA-GP and HPV-L1NK

    doi: 10.1101/2020.07.24.219329

    Figure Lengend Snippet: Expression efficiency of EBOV-GP in transfected CHOK1- and HEK-293 cells. Plasmids from Marburg (pCAGGS.cm5 + GP) and Heidelberg (pcDNA™6/V5-His + GP) were used for transfection of HEK-293 cells (3a), while the the plasmid pCDNA 3.1+ GP was used in CHO-K1 cells (3b). GP expression was detected by anti-ZEBOV GP mouse antibody (3B11), and anti-ZEBOV GP human antibody (KZ52). The binding to EBOV-GP transfected HEK-293-cells by the anti-GP mouse antibody 3B11 was trypsin-dependent (3a left). The human anti-GP did not show a dependency on trypsin (3a right). After transfection, the strongly positive cells were sorted and further checked for their degree of EBOV-GP expression by mAb anti EBOV-GP (KZ52).

    Article Snippet: P24 antigen capture assay Lentivirus particles based on the human immunodeficiency virus-1 (HIV-1) were produced in HEK-293 cells through transient transfection of 2 plasmids encoding components of the virus envelope.

    Techniques: Expressing, Transfection, Plasmid Preparation, Binding Assay

    NCRs, homing selectins and inhibitory Siglecs bind to HEK293 EBOV-GP cells: 4a. Analysis of the binding of different fusion proteins to HEK-293 EBOV-GP cells. The figure shows that Siglec 7, NKp44, and L-selectin-Fc have a higher affinity to the EBOV-GP transfected cells, while Nkp46, Siglec-3, P-selectin, siglec-5-Fc and CD24-Fc have an only slightly increased affinity than the respective controls. NKp30-Fc, E-selectin-Fc, NKG2D-Fc, and CD44-Fc have a slightly reduced affinity to the EBOV-GP transfected cells. The binding of PSGL-1-Fc, siglec-2-Fc, siglecs-4-Fc, siglec10-Fc, TIM-1-Fc and DC-SIGNR/DC-SIGN does not change. 4b: Examples are shown for strongly trypsin-dependent binding of some recombinant fusion proteins (e.g. selectins) to their co-partner on the surface of target cells. 4c: Transfection efficiency of two different HEK-293 EBOV-GP cells.

    Journal: bioRxiv

    Article Title: Activating NK- receptors, homing selectins and inhibitory Siglecs recognize EBOLA-GP and HPV-L1NK

    doi: 10.1101/2020.07.24.219329

    Figure Lengend Snippet: NCRs, homing selectins and inhibitory Siglecs bind to HEK293 EBOV-GP cells: 4a. Analysis of the binding of different fusion proteins to HEK-293 EBOV-GP cells. The figure shows that Siglec 7, NKp44, and L-selectin-Fc have a higher affinity to the EBOV-GP transfected cells, while Nkp46, Siglec-3, P-selectin, siglec-5-Fc and CD24-Fc have an only slightly increased affinity than the respective controls. NKp30-Fc, E-selectin-Fc, NKG2D-Fc, and CD44-Fc have a slightly reduced affinity to the EBOV-GP transfected cells. The binding of PSGL-1-Fc, siglec-2-Fc, siglecs-4-Fc, siglec10-Fc, TIM-1-Fc and DC-SIGNR/DC-SIGN does not change. 4b: Examples are shown for strongly trypsin-dependent binding of some recombinant fusion proteins (e.g. selectins) to their co-partner on the surface of target cells. 4c: Transfection efficiency of two different HEK-293 EBOV-GP cells.

    Article Snippet: P24 antigen capture assay Lentivirus particles based on the human immunodeficiency virus-1 (HIV-1) were produced in HEK-293 cells through transient transfection of 2 plasmids encoding components of the virus envelope.

    Techniques: Binding Assay, Transfection, Recombinant

    Immunostaining of HEK293 cells stably expressing EGFP-PICK1 alone or coexpressing WT, +Ala, 3xAla_618-620, or 3xAla_615-617 DAT and EGFP-PICK1. The cells were fixed and incubated with primary MAB369 anti-DAT antibody (1:1000), followed by secondary goat anti-rat Alexa Fluor 568 antibody and visualization by confocal fluorescence microscopy, as described in Materials and Methods. The anti-DAT (red) is shown on the left, the EGFP-PICK1 (green) is in the middle, and the overlay is on the right. Scale bar, 10 μm. All pictures have the same magnification.

    Journal: The Journal of Neuroscience

    Article Title: Surface Targeting of the Dopamine Transporter Involves Discrete Epitopes in the Distal C Terminus But Does Not Require Canonical PDZ Domain Interactions

    doi: 10.1523/JNEUROSCI.1863-04.2004

    Figure Lengend Snippet: Immunostaining of HEK293 cells stably expressing EGFP-PICK1 alone or coexpressing WT, +Ala, 3xAla_618-620, or 3xAla_615-617 DAT and EGFP-PICK1. The cells were fixed and incubated with primary MAB369 anti-DAT antibody (1:1000), followed by secondary goat anti-rat Alexa Fluor 568 antibody and visualization by confocal fluorescence microscopy, as described in Materials and Methods. The anti-DAT (red) is shown on the left, the EGFP-PICK1 (green) is in the middle, and the overlay is on the right. Scale bar, 10 μm. All pictures have the same magnification.

    Article Snippet: Membrane protein (10 μg), prepared from HEK293 cells as described above, was assayed in a total volume of 250 μl using a sodium phosphate buffer (50 m m Na2 HPO4 -NaH2 PO4 , pH 7.4) containing ∼0.25 n m [125 I]RTI-55 (PE Applied Biosystems, Foster City, CA) and increasing concentrations of competing nonlabeled RTI-55.

    Techniques: Immunostaining, Stable Transfection, Expressing, Incubation, Fluorescence, Microscopy

    Surface expression of WT and mutants transporters assessed by biotinylation. A , Surface biotinylation of HEK293 cells expressing WT, ΔLKV, β 2 -23, β 2 -3, or +Ala (left). Comparison of nontransfected cells with WT transfected cells (right). Surface biotinylated protein was purified using streptavidin beads and analyzed by SDS-PAGE, followed by immunoblotting with the M2 anti-FLAG antibody, as described in Materials and Methods. The surface-expressed hDAT migrated as a single glycosylated band corresponding to a molecular weight of ∼90 kDa. B , SDS-PAGE and immunoblotting with the M2 anti-FLAG antibody of total cellular protein (15 μg) from cells expressing WT, ΔLKV, β 2 -23, β 2 ). The gel shown is representative of at least three independent experiments.

    Journal: The Journal of Neuroscience

    Article Title: Surface Targeting of the Dopamine Transporter Involves Discrete Epitopes in the Distal C Terminus But Does Not Require Canonical PDZ Domain Interactions

    doi: 10.1523/JNEUROSCI.1863-04.2004

    Figure Lengend Snippet: Surface expression of WT and mutants transporters assessed by biotinylation. A , Surface biotinylation of HEK293 cells expressing WT, ΔLKV, β 2 -23, β 2 -3, or +Ala (left). Comparison of nontransfected cells with WT transfected cells (right). Surface biotinylated protein was purified using streptavidin beads and analyzed by SDS-PAGE, followed by immunoblotting with the M2 anti-FLAG antibody, as described in Materials and Methods. The surface-expressed hDAT migrated as a single glycosylated band corresponding to a molecular weight of ∼90 kDa. B , SDS-PAGE and immunoblotting with the M2 anti-FLAG antibody of total cellular protein (15 μg) from cells expressing WT, ΔLKV, β 2 -23, β 2 ). The gel shown is representative of at least three independent experiments.

    Article Snippet: Membrane protein (10 μg), prepared from HEK293 cells as described above, was assayed in a total volume of 250 μl using a sodium phosphate buffer (50 m m Na2 HPO4 -NaH2 PO4 , pH 7.4) containing ∼0.25 n m [125 I]RTI-55 (PE Applied Biosystems, Foster City, CA) and increasing concentrations of competing nonlabeled RTI-55.

    Techniques: Expressing, Transfection, Purification, SDS Page, Molecular Weight

    Effect of proteasome 26S inhibition on degradation of the DAT and ΔLKV in HEK293 cells. A , The ΔLKV and the WT ( B ) after incubation with MG132 for 0, 3, 6, or 9 hr, followed by incubation with PNGaseF or EndoH. Samples were analyzed by SDS-PAGE, followed by immunoblotting with the MAB369 anti-DAT antibody, as described in Materials and Methods. The fully mature N-glycosylated hDAT, which is sensitive to PNGaseF, eluted corresponding to a molecular weight of 90 kDa. The immature lyglycosylated form, which is sensitive to EndoH, eluted at ∼60 kDa. Complete deglycosylation by PNGaseF or EndoH results in elution of the DAT at ∼50 kDa. Protein from untransfected cells is included in the right lane as control.

    Journal: The Journal of Neuroscience

    Article Title: Surface Targeting of the Dopamine Transporter Involves Discrete Epitopes in the Distal C Terminus But Does Not Require Canonical PDZ Domain Interactions

    doi: 10.1523/JNEUROSCI.1863-04.2004

    Figure Lengend Snippet: Effect of proteasome 26S inhibition on degradation of the DAT and ΔLKV in HEK293 cells. A , The ΔLKV and the WT ( B ) after incubation with MG132 for 0, 3, 6, or 9 hr, followed by incubation with PNGaseF or EndoH. Samples were analyzed by SDS-PAGE, followed by immunoblotting with the MAB369 anti-DAT antibody, as described in Materials and Methods. The fully mature N-glycosylated hDAT, which is sensitive to PNGaseF, eluted corresponding to a molecular weight of 90 kDa. The immature lyglycosylated form, which is sensitive to EndoH, eluted at ∼60 kDa. Complete deglycosylation by PNGaseF or EndoH results in elution of the DAT at ∼50 kDa. Protein from untransfected cells is included in the right lane as control.

    Article Snippet: Membrane protein (10 μg), prepared from HEK293 cells as described above, was assayed in a total volume of 250 μl using a sodium phosphate buffer (50 m m Na2 HPO4 -NaH2 PO4 , pH 7.4) containing ∼0.25 n m [125 I]RTI-55 (PE Applied Biosystems, Foster City, CA) and increasing concentrations of competing nonlabeled RTI-55.

    Techniques: Inhibition, Incubation, SDS Page, Molecular Weight

    S534 phosphorylation affects DNA binding and gene expression by NF-κB at late time points through regulation of p65 stability ( A and B ) WT and S534A mice were injected i.v. with LPS (20 mg/kg) and sacrificed after the indicated times. (A) Liver tissue was analyzed by immunohistochemistry to monitor the translocation of p65 (red) to the nucleus (blue). (B) The percentages of cells with p65-positive nuclei were quantified. Data are means ± SD of five mice per genotype and time point. ( C ) Top: HEK 293 cells overexpressing human M2-p65 or M2-S536A-p65 were treated with TNF-α and then subjected to pulse-chase analysis for the indicated times to determine the half-life of p65 protein. Bottom: Data are means ± SD of three independent experiments, each performed in triplicate. ( D ) Top: WT and S534A MEFs were treated with cycloheximide (30 µg/ml) and then were left unstimulated or were stimulated with IL-1β for the indicated times. Samples were analyzed by Western blotting with antibodies against the indicated proteins. Bottom: The relative abundance of p65 protein, normalized to that of GAPDH, was determined for the indicated times by densitometric analysis. Data are means ± SD of three experiments. ( E ) Left: NF-κB DNA binding activity was determined in nuclear extracts from the livers of LPS-treated WT and S534A mice (n = 6 mice per genotype and time point). A value of 100 represents the positive control (recombinant human p65). The dashed line indicates NF-κB binding activity at baseline. Right: The competitive oligonucleotide suppressed DNA binding by the positive control. * P

    Journal: Science signaling

    Article Title: Negative regulation of NF-κB p65 activity by serine 536 phosphorylation

    doi: 10.1126/scisignal.aab2820

    Figure Lengend Snippet: S534 phosphorylation affects DNA binding and gene expression by NF-κB at late time points through regulation of p65 stability ( A and B ) WT and S534A mice were injected i.v. with LPS (20 mg/kg) and sacrificed after the indicated times. (A) Liver tissue was analyzed by immunohistochemistry to monitor the translocation of p65 (red) to the nucleus (blue). (B) The percentages of cells with p65-positive nuclei were quantified. Data are means ± SD of five mice per genotype and time point. ( C ) Top: HEK 293 cells overexpressing human M2-p65 or M2-S536A-p65 were treated with TNF-α and then subjected to pulse-chase analysis for the indicated times to determine the half-life of p65 protein. Bottom: Data are means ± SD of three independent experiments, each performed in triplicate. ( D ) Top: WT and S534A MEFs were treated with cycloheximide (30 µg/ml) and then were left unstimulated or were stimulated with IL-1β for the indicated times. Samples were analyzed by Western blotting with antibodies against the indicated proteins. Bottom: The relative abundance of p65 protein, normalized to that of GAPDH, was determined for the indicated times by densitometric analysis. Data are means ± SD of three experiments. ( E ) Left: NF-κB DNA binding activity was determined in nuclear extracts from the livers of LPS-treated WT and S534A mice (n = 6 mice per genotype and time point). A value of 100 represents the positive control (recombinant human p65). The dashed line indicates NF-κB binding activity at baseline. Right: The competitive oligonucleotide suppressed DNA binding by the positive control. * P

    Article Snippet: Human embryonic kidney (HEK) 293 cells were transfected with lipofectamine (Life Technologies) with plasmids encoding human M2-p65 or M2-S536A-p65.

    Techniques: Binding Assay, Expressing, Mouse Assay, Injection, Immunohistochemistry, Translocation Assay, Pulse Chase, Western Blot, Activity Assay, Positive Control, Recombinant

    Glycosylation on the uncleaved, soluble NFL Env trimer. a ) illustrates the protection by the bound antibodies from EndoH trimming, and the glycan fence around the PGV19 footprint (LC var : cyan, HC var : orange). On the right are various conformations of well-defined glycan sub- or super-structures observed in the BG505 NFL.664 crystal structure. b HILIC-UPLC spectra of fluorescently labeled N-linked glycans released from BG505 NFL.664 and BG505 SOSIP.664 produced in HEK 293F cells. Oligomannose and hybrid glycans are colored in green and complex glycans in pink. The corresponding structures for the oligomannose glycans (Man 5-9 GlcNAc 2 ) are shown in the upper panel. c Site-specific N-glycosylation analysis of BG505 NFL.664. Relative quantification of the micro-heterogeneity of 20 out of the 27 N-glycosylation sites of BG505 NFL.664 produced in HEK 293F cells were determined, with the color scheme preserved from panel b . Glycoform annotation: oligomannose glycans Man 5 GlcNAc 2 to Man 9 GlcNAc 2 are represented as M5 to M9, hybrids as H, and fucosylated hybrids as FH. Complex glycans are also categorized by the number of branching antennae (A n ); number of galactose residues (G n ); and core fucose (f)

    Journal: Nature Communications

    Article Title: Structure of a cleavage-independent HIV Env recapitulates the glycoprotein architecture of the native cleaved trimer

    doi: 10.1038/s41467-018-04272-y

    Figure Lengend Snippet: Glycosylation on the uncleaved, soluble NFL Env trimer. a ) illustrates the protection by the bound antibodies from EndoH trimming, and the glycan fence around the PGV19 footprint (LC var : cyan, HC var : orange). On the right are various conformations of well-defined glycan sub- or super-structures observed in the BG505 NFL.664 crystal structure. b HILIC-UPLC spectra of fluorescently labeled N-linked glycans released from BG505 NFL.664 and BG505 SOSIP.664 produced in HEK 293F cells. Oligomannose and hybrid glycans are colored in green and complex glycans in pink. The corresponding structures for the oligomannose glycans (Man 5-9 GlcNAc 2 ) are shown in the upper panel. c Site-specific N-glycosylation analysis of BG505 NFL.664. Relative quantification of the micro-heterogeneity of 20 out of the 27 N-glycosylation sites of BG505 NFL.664 produced in HEK 293F cells were determined, with the color scheme preserved from panel b . Glycoform annotation: oligomannose glycans Man 5 GlcNAc 2 to Man 9 GlcNAc 2 are represented as M5 to M9, hybrids as H, and fucosylated hybrids as FH. Complex glycans are also categorized by the number of branching antennae (A n ); number of galactose residues (G n ); and core fucose (f)

    Article Snippet: Briefly, HEK 293F cells (Life Technologies catalog# ,±10 mg swainsonine/2.5 mg kifunensine) were transfected with plasmids encoding soluble BG505 NFL.664 trimer protein (GenScript).

    Techniques: Hydrophilic Interaction Liquid Chromatography, Labeling, Produced

    Biophysical characterization of sequential binding events of bnAbs with the NFL Env trimer. The top panel illustrates binding by ITC of PGT122 and PGV19 individually and then sequentially to BG505 NFL.664 produced in HEK 293F cells to which kifunensine and swainsonine were added (resulting in oligomannose glycoforms only) and depicted within the green outlined box). The top right illustration highlights the location of PGT122 and PGV19 at the N332-supersite and the CD4bs, respectively, on one protomer as a colored cartoon. The three protomers of the Env trimer are shown in separate colors in surface representation. The second panel shows the binding of the individual antibodies and then their successive binding to BG505 NFL.664 produced in HEK293F cells (comprising of oligomannose, complex and hybrid glycans as detected by mass spectrometry studies, outlined by a pink box). The middle illustration on the right shows different glycoforms modeled on N301 (Man 9 : green, complex glycan: magenta) and the location of PGV19 and PGT122 as viewed from the trimer apex. The third panel describes the effect of the removal of N276 glycan on the binding of PGT122 and PGV19 individually, followed by the sequential binding of both antibodies to the N276D mutant. The bottom right illustration shows the distance between the variable regions of PGV19 and PGT122 on adjacent protomers. All values of K d are nanomolar, enthalpy change (∆ H ) is cal/mol, and entropy (∆ S ) is cal/mol/deg. Binding stoichiometry ( N ) is directly affected by uncertainties in protein concentration measurement, total active molecules in the sample, and glycan heterogeneity. Associated errors/uncertainties are less than 10% of the average of at least two independent binding measurements

    Journal: Nature Communications

    Article Title: Structure of a cleavage-independent HIV Env recapitulates the glycoprotein architecture of the native cleaved trimer

    doi: 10.1038/s41467-018-04272-y

    Figure Lengend Snippet: Biophysical characterization of sequential binding events of bnAbs with the NFL Env trimer. The top panel illustrates binding by ITC of PGT122 and PGV19 individually and then sequentially to BG505 NFL.664 produced in HEK 293F cells to which kifunensine and swainsonine were added (resulting in oligomannose glycoforms only) and depicted within the green outlined box). The top right illustration highlights the location of PGT122 and PGV19 at the N332-supersite and the CD4bs, respectively, on one protomer as a colored cartoon. The three protomers of the Env trimer are shown in separate colors in surface representation. The second panel shows the binding of the individual antibodies and then their successive binding to BG505 NFL.664 produced in HEK293F cells (comprising of oligomannose, complex and hybrid glycans as detected by mass spectrometry studies, outlined by a pink box). The middle illustration on the right shows different glycoforms modeled on N301 (Man 9 : green, complex glycan: magenta) and the location of PGV19 and PGT122 as viewed from the trimer apex. The third panel describes the effect of the removal of N276 glycan on the binding of PGT122 and PGV19 individually, followed by the sequential binding of both antibodies to the N276D mutant. The bottom right illustration shows the distance between the variable regions of PGV19 and PGT122 on adjacent protomers. All values of K d are nanomolar, enthalpy change (∆ H ) is cal/mol, and entropy (∆ S ) is cal/mol/deg. Binding stoichiometry ( N ) is directly affected by uncertainties in protein concentration measurement, total active molecules in the sample, and glycan heterogeneity. Associated errors/uncertainties are less than 10% of the average of at least two independent binding measurements

    Article Snippet: Briefly, HEK 293F cells (Life Technologies catalog# ,±10 mg swainsonine/2.5 mg kifunensine) were transfected with plasmids encoding soluble BG505 NFL.664 trimer protein (GenScript).

    Techniques: Binding Assay, Produced, Mass Spectrometry, Mutagenesis, Protein Concentration

    (A and B) 1-CP-U-induced cytotoxicity. A total of 5,000 cells were seeded in triplicate in 96-well plates and then incubated with 1-CP-U for five days. The LS174T, CaSki, MRC-5 and HEK-293 cells were subject to treatment with 1-CP-U (1.0 μmol/l). The SKOV3, HeLa, SMMC-7721 and A549 cells were treated with increasing concentrations of 1-CP-U (0.7, 1.0 and 1.4 μmol/l). The cell viability was then analyzed by MTT assay. Data are presented as the mean ± standard deviation of three separate experiments. 1-CP-U, 1-calcium phosphate-uracil.

    Journal: Molecular Medicine Reports

    Article Title: 1-calcium phosphate-uracil, a synthesized pyrimidine derivative agent, has anti-proliferative, pro-apoptotic and anti-invasion effects on multiple tumor cell lines

    doi: 10.3892/mmr.2014.2489

    Figure Lengend Snippet: (A and B) 1-CP-U-induced cytotoxicity. A total of 5,000 cells were seeded in triplicate in 96-well plates and then incubated with 1-CP-U for five days. The LS174T, CaSki, MRC-5 and HEK-293 cells were subject to treatment with 1-CP-U (1.0 μmol/l). The SKOV3, HeLa, SMMC-7721 and A549 cells were treated with increasing concentrations of 1-CP-U (0.7, 1.0 and 1.4 μmol/l). The cell viability was then analyzed by MTT assay. Data are presented as the mean ± standard deviation of three separate experiments. 1-CP-U, 1-calcium phosphate-uracil.

    Article Snippet: Cell lines and culture Human cervical cancer cell lines HeLa and CaSki, human ovarian cancer cell line SKOV3, human hepatocellular carcinoma cell line SMMC-7721, human lung adenocarcinoma cell line A549, human colorectal carcinoma cell line LS174T, normal lung fibroblasts MRC-5 and human embryonic kidney (HEK-293) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Incubation, MTT Assay, Standard Deviation

    Effects of AngII stimulation on the distribution of MP-YFP, PLCδ1-PH-YFP, and KR-YFP in HEK293 cells. The cells were transfected with the AT 1 -R-luciferase and with the indicated YFP-fused proteins. After 24 h, the cells were exposed to 100 n m

    Journal: The Journal of Biological Chemistry

    Article Title: Mapping of the Localization of Type 1 Angiotensin Receptor in Membrane Microdomains Using Bioluminescence Resonance Energy Transfer-based Sensors

    doi: 10.1074/jbc.M111.293944

    Figure Lengend Snippet: Effects of AngII stimulation on the distribution of MP-YFP, PLCδ1-PH-YFP, and KR-YFP in HEK293 cells. The cells were transfected with the AT 1 -R-luciferase and with the indicated YFP-fused proteins. After 24 h, the cells were exposed to 100 n m

    Article Snippet: The human embryonic kidney (HEK293) cells were from ATCC (American Type Culture Collection, Manassas, VA).

    Techniques: Transfection, Luciferase

    BRET assay between AT 1 -R and different proteins upon AngII stimulation in HEK293 cells. HEK293 cells were transfected with the plasmids of the indicated AT 1 -R-luciferase ( magenta trace , wild type; green trace , DRY/AAY mutant; blue trace , TSTS/A mutant)

    Journal: The Journal of Biological Chemistry

    Article Title: Mapping of the Localization of Type 1 Angiotensin Receptor in Membrane Microdomains Using Bioluminescence Resonance Energy Transfer-based Sensors

    doi: 10.1074/jbc.M111.293944

    Figure Lengend Snippet: BRET assay between AT 1 -R and different proteins upon AngII stimulation in HEK293 cells. HEK293 cells were transfected with the plasmids of the indicated AT 1 -R-luciferase ( magenta trace , wild type; green trace , DRY/AAY mutant; blue trace , TSTS/A mutant)

    Article Snippet: The human embryonic kidney (HEK293) cells were from ATCC (American Type Culture Collection, Manassas, VA).

    Techniques: Bioluminescence Resonance Energy Transfer, Transfection, Luciferase, Mutagenesis

    Effects of AngII and candesartan on the BRET interaction between AT 1 -R and different proteins in HEK293 cells. HEK293 cells were transfected with the plasmids of the AT 1 -R-luciferase and the indicated YFP-fused proteins, and after 24 h, the cells were

    Journal: The Journal of Biological Chemistry

    Article Title: Mapping of the Localization of Type 1 Angiotensin Receptor in Membrane Microdomains Using Bioluminescence Resonance Energy Transfer-based Sensors

    doi: 10.1074/jbc.M111.293944

    Figure Lengend Snippet: Effects of AngII and candesartan on the BRET interaction between AT 1 -R and different proteins in HEK293 cells. HEK293 cells were transfected with the plasmids of the AT 1 -R-luciferase and the indicated YFP-fused proteins, and after 24 h, the cells were

    Article Snippet: The human embryonic kidney (HEK293) cells were from ATCC (American Type Culture Collection, Manassas, VA).

    Techniques: Bioluminescence Resonance Energy Transfer, Transfection, Luciferase

    Effects of angiotensin peptides on the BRET interaction between AT 1 -R and different proteins in HEK293 cells. HEK293 cells were transfected with the plasmids of the AT 1 -R-luciferase and the indicated YFP-fused proteins, and after 24 h, the cells were

    Journal: The Journal of Biological Chemistry

    Article Title: Mapping of the Localization of Type 1 Angiotensin Receptor in Membrane Microdomains Using Bioluminescence Resonance Energy Transfer-based Sensors

    doi: 10.1074/jbc.M111.293944

    Figure Lengend Snippet: Effects of angiotensin peptides on the BRET interaction between AT 1 -R and different proteins in HEK293 cells. HEK293 cells were transfected with the plasmids of the AT 1 -R-luciferase and the indicated YFP-fused proteins, and after 24 h, the cells were

    Article Snippet: The human embryonic kidney (HEK293) cells were from ATCC (American Type Culture Collection, Manassas, VA).

    Techniques: Bioluminescence Resonance Energy Transfer, Transfection, Luciferase

    HOTAIRM1 regulates autophagy-associated gene expression by competing with the binding sites of miRNAs. ( a ) HOTAIRM1 transcripts bind to AGO2 directly. AGO2 was assayed by western blotting after acquiring the possible protein complex binding to HOTAIRM1. Proteins bound to the antisense of HOTAIRM1 served as loading controls. ( b ) RNA immunoprecipitation was performed to acquire the RNA that interacted with AGO2 protein. The qRT-PCR product of HOTAIRM1 was tested by agarose gel electrophoresis. ( c ) Luciferase reporter assays analyzing the binding of HOTAIRM1 to miRNAs. NC and miRNAs duplexes were co-transfected with psiCHECH-2 plasmids containing the 59 nt of HOTAIRM1-WT or HOTAIRM1-MUT. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity ( n =3 independent experiments performed in triplicate). ( d ) Luciferase reporter assays analyzing the genes potentially influenced by HOTAIRM1. NC and two HOTAIRM1-siRNAs were co-transfected with psiCHECH-2 plasmids with the 59-nt of miRNAs targeting the wild-type or mutant gene position in HEK-293 T cells. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. Values are derived from  n =3 independent experiments, and data are reported as the mean±S.D. ( e ) Western blotting detecting the proteins expression levels of genes regulated by HOTAIRM1. ULK1, E2F1 and DRAM2 were all downregulated when HOTAIRM1 was knocked down in NB4 cells with or without 1  μ M ATRA treatment; GAPDH was used as a loading control. The densitometric ratio normalized to GAPDH was recorded by quantity one. Values are derived from  n =3 independent experiments, and data are reported as the mean±S.D. (down). ( f–h ) The qRT-PCR testing of DRMA1, LC3B and ULK1, three direct targets of E2F1. Experiments were performed in triplicate and are reported as the mean±S.D. ( i ) The qRT-PCR testing of miRNAs in NB4-Lv-NC and NB4-Lv-shHOTAIRM1 cells after treatment with ATRA (1  μ M, 48 h). Experiments were performed in triplicate and normalized to GAPDH. ( j ) The detection of PML-RARA by western bloting after transient overexpression of miR-20a and miR-106b mimics in NB4 cells. Experiments were performed in triplicate. PML-RARA/GAPDH densitometric ratios were recorded. Differences in  c - i  were considered significant at * P

    Journal: Cell Death and Differentiation

    Article Title: The lncRNA HOTAIRM1 regulates the degradation of PML-RARA oncoprotein and myeloid cell differentiation by enhancing the autophagy pathway

    doi: 10.1038/cdd.2016.111

    Figure Lengend Snippet: HOTAIRM1 regulates autophagy-associated gene expression by competing with the binding sites of miRNAs. ( a ) HOTAIRM1 transcripts bind to AGO2 directly. AGO2 was assayed by western blotting after acquiring the possible protein complex binding to HOTAIRM1. Proteins bound to the antisense of HOTAIRM1 served as loading controls. ( b ) RNA immunoprecipitation was performed to acquire the RNA that interacted with AGO2 protein. The qRT-PCR product of HOTAIRM1 was tested by agarose gel electrophoresis. ( c ) Luciferase reporter assays analyzing the binding of HOTAIRM1 to miRNAs. NC and miRNAs duplexes were co-transfected with psiCHECH-2 plasmids containing the 59 nt of HOTAIRM1-WT or HOTAIRM1-MUT. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity ( n =3 independent experiments performed in triplicate). ( d ) Luciferase reporter assays analyzing the genes potentially influenced by HOTAIRM1. NC and two HOTAIRM1-siRNAs were co-transfected with psiCHECH-2 plasmids with the 59-nt of miRNAs targeting the wild-type or mutant gene position in HEK-293 T cells. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. Values are derived from n =3 independent experiments, and data are reported as the mean±S.D. ( e ) Western blotting detecting the proteins expression levels of genes regulated by HOTAIRM1. ULK1, E2F1 and DRAM2 were all downregulated when HOTAIRM1 was knocked down in NB4 cells with or without 1  μ M ATRA treatment; GAPDH was used as a loading control. The densitometric ratio normalized to GAPDH was recorded by quantity one. Values are derived from n =3 independent experiments, and data are reported as the mean±S.D. (down). ( f–h ) The qRT-PCR testing of DRMA1, LC3B and ULK1, three direct targets of E2F1. Experiments were performed in triplicate and are reported as the mean±S.D. ( i ) The qRT-PCR testing of miRNAs in NB4-Lv-NC and NB4-Lv-shHOTAIRM1 cells after treatment with ATRA (1  μ M, 48 h). Experiments were performed in triplicate and normalized to GAPDH. ( j ) The detection of PML-RARA by western bloting after transient overexpression of miR-20a and miR-106b mimics in NB4 cells. Experiments were performed in triplicate. PML-RARA/GAPDH densitometric ratios were recorded. Differences in c - i were considered significant at * P

    Article Snippet: Cell lines and cultures Leukemia cells (NB4 and U937-PR9cells) and HEK-293 T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA.) and were cultured in RPMI Medium Modified (HyClone, South Logan, UT, USA) and DMEM (HyClone), respectively, containing 10% fetal bovine serum (Gibco, ThermoFisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Binding Assay, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Luciferase, Transfection, Activity Assay, Mutagenesis, Derivative Assay, Over Expression

    Macroscopic properties of expressed and native ClC-1 currents. Voltage dependence of average instantaneous (open symbols) and steady-state (closed symbols) ClC-1 currents recorded from mClC-1–expressing HEK293 cells (A; n = 12), mClC-1–expressing skeletal myotubes (B; n = 4), and native FDB fibers obtained from 18–20-d-old WT mice (C; n = 8). Instantaneous currents measured in control noninjected myotubes did not display appreciable ClC-1 currents (upright triangles). (D) Superimposed and normalized instantaneous current–voltage relationships obtained from mClC-1– expressing HEK293 cells (squares), myotubes (inverted triangles), and native WT FDB fibers (circles). (E) Average relative Po - V curves for mClC-1–expressing HEK293 cells (squares), myotubes (inverted triangles), and native WT FDB fibers (circles) obtained from tail currents elicited at −100 mV. Smooth curves through each P O - V dataset were generated using a modified Boltzmann equation ( Eq. 3 ).

    Journal: The Journal of General Physiology

    Article Title: Muscle Chloride Channel Dysfunction in Two Mouse Models of Myotonic Dystrophy

    doi: 10.1085/jgp.200609635

    Figure Lengend Snippet: Macroscopic properties of expressed and native ClC-1 currents. Voltage dependence of average instantaneous (open symbols) and steady-state (closed symbols) ClC-1 currents recorded from mClC-1–expressing HEK293 cells (A; n = 12), mClC-1–expressing skeletal myotubes (B; n = 4), and native FDB fibers obtained from 18–20-d-old WT mice (C; n = 8). Instantaneous currents measured in control noninjected myotubes did not display appreciable ClC-1 currents (upright triangles). (D) Superimposed and normalized instantaneous current–voltage relationships obtained from mClC-1– expressing HEK293 cells (squares), myotubes (inverted triangles), and native WT FDB fibers (circles). (E) Average relative Po - V curves for mClC-1–expressing HEK293 cells (squares), myotubes (inverted triangles), and native WT FDB fibers (circles) obtained from tail currents elicited at −100 mV. Smooth curves through each P O - V dataset were generated using a modified Boltzmann equation ( Eq. 3 ).

    Article Snippet: Thus, comparing mClC-1 properties after heterologous expression in HEK293 cells with that observed in native FDB fibers and after homologous expression in myotubes could reveal potential “muscle-specific” modulatory effects on ClC-1 function that may not be faithfully reproduced using standard heterologous expression systems (e.g., HEK293 cells).

    Techniques: Expressing, Mouse Assay, Generated, Modification

    Deactivation kinetics of expressed and native ClC-1 channels. Voltage dependence for the relative contributions of the slow (A), fast (B), and nondeactivating (C) gating components of ClC-1 deactivation in mClC-1–expressing HEK293 cells (squares; n = 12), mClC-1–expressing myotubes (triangles; n = 4), and native FDB fibers obtained from 18–20-d-old WT mice (circles; n = 8). (D) Voltage dependence of the time constants for the fast (lower symbols) and slow (upper symbols) components of ClC-1 deactivation. *, P ≤ 0.05 HEK293 cells compared with WT FDB fibers.

    Journal: The Journal of General Physiology

    Article Title: Muscle Chloride Channel Dysfunction in Two Mouse Models of Myotonic Dystrophy

    doi: 10.1085/jgp.200609635

    Figure Lengend Snippet: Deactivation kinetics of expressed and native ClC-1 channels. Voltage dependence for the relative contributions of the slow (A), fast (B), and nondeactivating (C) gating components of ClC-1 deactivation in mClC-1–expressing HEK293 cells (squares; n = 12), mClC-1–expressing myotubes (triangles; n = 4), and native FDB fibers obtained from 18–20-d-old WT mice (circles; n = 8). (D) Voltage dependence of the time constants for the fast (lower symbols) and slow (upper symbols) components of ClC-1 deactivation. *, P ≤ 0.05 HEK293 cells compared with WT FDB fibers.

    Article Snippet: Thus, comparing mClC-1 properties after heterologous expression in HEK293 cells with that observed in native FDB fibers and after homologous expression in myotubes could reveal potential “muscle-specific” modulatory effects on ClC-1 function that may not be faithfully reproduced using standard heterologous expression systems (e.g., HEK293 cells).

    Techniques: Expressing, Mouse Assay

    Chloride channel currents from mClC-1–expressing HEK293 cells, mClC-1–expressing mouse skeletal myotubes, and native FDB fibers obtained from 18–20-d-old WT mice. Representative whole-cell currents recorded first in the absence (Raw) and then in the presence of 1 mM 9AC (9AC) using an identical voltage protocol (top). ClC-1 currents were then quantified after offline subtraction of 9AC-insensitive currents from raw currents (9AC sensitive). (Inset) Capacitative current recorded from the WT FDB fiber resulting from the average of five 10-mV depolarizations delivered from a −80-mV holding potential. The time constant the capacitative current relaxation was fitted (thick line) to a first-order exponential function (τ m = 339 μs). Cell capacitance and access resistance deduced from the fit were 671 pF and 509 kΩ, respectively. The scale bars for the inset are 6 nA (vertical) and 2 ms (horizontal). The dashed lines represent the zero current level.

    Journal: The Journal of General Physiology

    Article Title: Muscle Chloride Channel Dysfunction in Two Mouse Models of Myotonic Dystrophy

    doi: 10.1085/jgp.200609635

    Figure Lengend Snippet: Chloride channel currents from mClC-1–expressing HEK293 cells, mClC-1–expressing mouse skeletal myotubes, and native FDB fibers obtained from 18–20-d-old WT mice. Representative whole-cell currents recorded first in the absence (Raw) and then in the presence of 1 mM 9AC (9AC) using an identical voltage protocol (top). ClC-1 currents were then quantified after offline subtraction of 9AC-insensitive currents from raw currents (9AC sensitive). (Inset) Capacitative current recorded from the WT FDB fiber resulting from the average of five 10-mV depolarizations delivered from a −80-mV holding potential. The time constant the capacitative current relaxation was fitted (thick line) to a first-order exponential function (τ m = 339 μs). Cell capacitance and access resistance deduced from the fit were 671 pF and 509 kΩ, respectively. The scale bars for the inset are 6 nA (vertical) and 2 ms (horizontal). The dashed lines represent the zero current level.

    Article Snippet: Thus, comparing mClC-1 properties after heterologous expression in HEK293 cells with that observed in native FDB fibers and after homologous expression in myotubes could reveal potential “muscle-specific” modulatory effects on ClC-1 function that may not be faithfully reproduced using standard heterologous expression systems (e.g., HEK293 cells).

    Techniques: Expressing, Mouse Assay, Mass Spectrometry

    Utilization of pMVP for the creation of unique PDX1 vectors. pENTR plasmids containing cDNA for human PDX1 with or without a stop codon (i.e. open) were recombined with pMVP components to generate an assortment of PDX1-expressing ( A ) adenovirus and ( C ) expression plasmid vectors. ( B ) Immunoblot blot analysis of INS1 832/13 cell lysates harvested 48h after treatment with crude adenovirus lysates. For the epitope tagged conditions, note the appearance of the endogenous (lower) and overexpressed (upper) PDX1 bands (top blot). For eGFP blot, the use of P2A adds 23 amino acids to the N-terminal protein (i.e. eGFP). Immunoblot analysis of HEK293 cell lysates 24 h after transfection of expression vectors encoding PDX1 with C-terminal ( D ) epitope tags, ( E ) eGFP reporter or fusion, ( G ) and mCherry reporter or fusion. ( F, H ) Fluorescence microscopy of the eGFP and mCherry containing conditions analyzed in panels E and G. Visible bands resulting from ‘Uncleaved’ P2A products (red circle •) and degradation products (*) are labeled.

    Journal: Nucleic Acids Research

    Article Title: Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing

    doi: 10.1093/nar/gky1286

    Figure Lengend Snippet: Utilization of pMVP for the creation of unique PDX1 vectors. pENTR plasmids containing cDNA for human PDX1 with or without a stop codon (i.e. open) were recombined with pMVP components to generate an assortment of PDX1-expressing ( A ) adenovirus and ( C ) expression plasmid vectors. ( B ) Immunoblot blot analysis of INS1 832/13 cell lysates harvested 48h after treatment with crude adenovirus lysates. For the epitope tagged conditions, note the appearance of the endogenous (lower) and overexpressed (upper) PDX1 bands (top blot). For eGFP blot, the use of P2A adds 23 amino acids to the N-terminal protein (i.e. eGFP). Immunoblot analysis of HEK293 cell lysates 24 h after transfection of expression vectors encoding PDX1 with C-terminal ( D ) epitope tags, ( E ) eGFP reporter or fusion, ( G ) and mCherry reporter or fusion. ( F, H ) Fluorescence microscopy of the eGFP and mCherry containing conditions analyzed in panels E and G. Visible bands resulting from ‘Uncleaved’ P2A products (red circle •) and degradation products (*) are labeled.

    Article Snippet: For purification, six confluent 15 cm plates of HEK293 cells were transduced with secondary lysate and harvested after 40–60 h, at a point when 50–75% of cells were floating.

    Techniques: Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Labeling

    The pMAGIC toolbox. ( A ) A schematic representation of the structure of pMAGIC-derived single, double, and triple gRNA expressing vectors. Color scheme of the components matches the respective vector combinations. ( B ) Sa-dCas9 and Sa-Cas9 pMAGIC fusion genes cloned into pENTR221 R4-R3 (yellow). Additional effectors can be fused to Sa-dCas9 through unique NcoI (N-terminal) and BamHI (C-terminus) restriction sites. ( C ) pENTR221 L1-R5 plasmids (purple) containing a Sa-Cas9 gRNA expression cassette driven by the mouse U6 (mU6) or human U6 (U6) promoter. Alternatively, a U6-driven Sa-Cas9 gRNA expression cassette can be combined with a CMV, EF1a, or RIP promoter. ( D ) pENTR221 L5-L4 plasmids (blue) containing a U6-driven Sa-Cas9 gRNA expression cassette and CMV, EF1a, or RIP promoter. ( E ) pENTR221 L3-L2 plasmid (red) containing a 3xHA epitope tag for C-terminal fusion to Sa-Cas9 proteins and a U6-driven Sa-Cas9 gRNA expression cassette. (C–E) Protospacer oligonucleotides can be ligated into BsaI digested gRNA expression cassettes. ( F ) Sa-Cas9 and x-Cas9 gRNA binding sites within the TRE promoter. Schematic of the TRE-driven eGFP reporter used to monitor dCas9-mediated transcriptional activation. ( G ) Schematic of mCherry-P2A-dCas9 expression vectors. HEK293 cells were co-transfected with plasmids for the eGFP reporter shown in panel (F), equimolar amounts of the indicated mCherry-P2A-dCas9 vectors, and the corresponding Sa-Cas9 gRNA TRE#1 or x-Cas9 gRNA TRE #1 plasmids. 72h after transfection, cells were ( H ) visualized by fluorescence microscopy and ( I ) cellular lysates were analyzed by immunoblot. ‘Uncleaved’ P2A products from Sa-dCas9 and x-dCas9 are designated by a red circle (•).

    Journal: Nucleic Acids Research

    Article Title: Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing

    doi: 10.1093/nar/gky1286

    Figure Lengend Snippet: The pMAGIC toolbox. ( A ) A schematic representation of the structure of pMAGIC-derived single, double, and triple gRNA expressing vectors. Color scheme of the components matches the respective vector combinations. ( B ) Sa-dCas9 and Sa-Cas9 pMAGIC fusion genes cloned into pENTR221 R4-R3 (yellow). Additional effectors can be fused to Sa-dCas9 through unique NcoI (N-terminal) and BamHI (C-terminus) restriction sites. ( C ) pENTR221 L1-R5 plasmids (purple) containing a Sa-Cas9 gRNA expression cassette driven by the mouse U6 (mU6) or human U6 (U6) promoter. Alternatively, a U6-driven Sa-Cas9 gRNA expression cassette can be combined with a CMV, EF1a, or RIP promoter. ( D ) pENTR221 L5-L4 plasmids (blue) containing a U6-driven Sa-Cas9 gRNA expression cassette and CMV, EF1a, or RIP promoter. ( E ) pENTR221 L3-L2 plasmid (red) containing a 3xHA epitope tag for C-terminal fusion to Sa-Cas9 proteins and a U6-driven Sa-Cas9 gRNA expression cassette. (C–E) Protospacer oligonucleotides can be ligated into BsaI digested gRNA expression cassettes. ( F ) Sa-Cas9 and x-Cas9 gRNA binding sites within the TRE promoter. Schematic of the TRE-driven eGFP reporter used to monitor dCas9-mediated transcriptional activation. ( G ) Schematic of mCherry-P2A-dCas9 expression vectors. HEK293 cells were co-transfected with plasmids for the eGFP reporter shown in panel (F), equimolar amounts of the indicated mCherry-P2A-dCas9 vectors, and the corresponding Sa-Cas9 gRNA TRE#1 or x-Cas9 gRNA TRE #1 plasmids. 72h after transfection, cells were ( H ) visualized by fluorescence microscopy and ( I ) cellular lysates were analyzed by immunoblot. ‘Uncleaved’ P2A products from Sa-dCas9 and x-dCas9 are designated by a red circle (•).

    Article Snippet: For purification, six confluent 15 cm plates of HEK293 cells were transduced with secondary lysate and harvested after 40–60 h, at a point when 50–75% of cells were floating.

    Techniques: Derivative Assay, Expressing, Plasmid Preparation, Clone Assay, Binding Assay, Activation Assay, Transfection, Fluorescence, Microscopy

    Larger deletions within the stalk region abrogate ADAM10 and ADAM17 mediated shedding. HEK293 cells were transfected with expression plasmids encoding B IL-6RΔA333_V362 ( A ), IL-6RΔE317_T352 ( C and D ), IL-6RΔA323_V362 ( E and F ),

    Journal: The Journal of Biological Chemistry

    Article Title: Minimal Interleukin 6 (IL-6) Receptor Stalk Composition for IL-6 Receptor Shedding and IL-6 Classic Signaling

    doi: 10.1074/jbc.M113.466169

    Figure Lengend Snippet: Larger deletions within the stalk region abrogate ADAM10 and ADAM17 mediated shedding. HEK293 cells were transfected with expression plasmids encoding B IL-6RΔA333_V362 ( A ), IL-6RΔE317_T352 ( C and D ), IL-6RΔA323_V362 ( E and F ),

    Article Snippet: Shortening of the IL-6R stalk by 10 amino acids does not abrogate ADAM17 substrate recognition as long as the cleavage site is retained, but again, an overall reduction of the total amount of shed IL-6R compared with wild type IL-6R to 52.4 ± 19.3% (IL-6RΔI343_T352) and 63.8 ± 29.2% (IL-6RΔA333_N342) was detected in HEK293 cells.

    Techniques: Transfection, Expressing

    Deletions within the stalk region differentially influence constitutive IL-6R shedding. A and B , HEK293 cells were transfected with the indicated IL-6R constructs. 48 h after transfection medium was replaced with serum-free medium. The amount of constitutive

    Journal: The Journal of Biological Chemistry

    Article Title: Minimal Interleukin 6 (IL-6) Receptor Stalk Composition for IL-6 Receptor Shedding and IL-6 Classic Signaling

    doi: 10.1074/jbc.M113.466169

    Figure Lengend Snippet: Deletions within the stalk region differentially influence constitutive IL-6R shedding. A and B , HEK293 cells were transfected with the indicated IL-6R constructs. 48 h after transfection medium was replaced with serum-free medium. The amount of constitutive

    Article Snippet: Shortening of the IL-6R stalk by 10 amino acids does not abrogate ADAM17 substrate recognition as long as the cleavage site is retained, but again, an overall reduction of the total amount of shed IL-6R compared with wild type IL-6R to 52.4 ± 19.3% (IL-6RΔI343_T352) and 63.8 ± 29.2% (IL-6RΔA333_N342) was detected in HEK293 cells.

    Techniques: Transfection, Construct

    Mean luciferase activity of HEK293 TLR reporter cell lines incubated with different fibers (at approx. 400 μg/mL) and positive controls [TNF (5 ng/mL), Pam3-CSK 4 (200 ng/mL), and LPS (250 ng/mL)]. Error bars represent the standard error of the mean values. Asterisks represent classes of statistically significant different responses compared to the medium control (* P

    Journal: Frontiers in Immunology

    Article Title: Structure Dependent-Immunomodulation by Sugar Beet Arabinans via a SYK Tyrosine Kinase-Dependent Signaling Pathway

    doi: 10.3389/fimmu.2018.01972

    Figure Lengend Snippet: Mean luciferase activity of HEK293 TLR reporter cell lines incubated with different fibers (at approx. 400 μg/mL) and positive controls [TNF (5 ng/mL), Pam3-CSK 4 (200 ng/mL), and LPS (250 ng/mL)]. Error bars represent the standard error of the mean values. Asterisks represent classes of statistically significant different responses compared to the medium control (* P

    Article Snippet: Briefly, HEK293 cells were transfected with one of the mentioned human TLR(s) and pNiFty-luc, a NF-κB luciferase reporter construct (Invivogen, Toulouse, France, catalog numbers 293-htlr2; 293-htlr4a; 293-htlr5; 293-htlr2/6; 293-mtlr1/2; pnifty-luc).

    Techniques: Luciferase, Activity Assay, Incubation

    PP2A activity is depressed in vivo by expression of pseudophosphorylated B56α in HEK293 cells. A , protein phosphatase ( PP ) activity was determined in reaction mixtures containing either purified heterodimeric PP2A CA or homogenized HEK293 cells

    Journal: The Journal of Biological Chemistry

    Article Title: Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41 *

    doi: 10.1074/jbc.M113.507996

    Figure Lengend Snippet: PP2A activity is depressed in vivo by expression of pseudophosphorylated B56α in HEK293 cells. A , protein phosphatase ( PP ) activity was determined in reaction mixtures containing either purified heterodimeric PP2A CA or homogenized HEK293 cells

    Article Snippet: Cultured HEK293 cells (see above) were maintained on 24-well plates (Corning Glass) and transfected using TurboFectTM in vitro transfection reagent (Fermentas) according to manufacturer's protocol.

    Techniques: Activity Assay, In Vivo, Expressing, Purification

    Expression of GFP-tagged B56α in HEK293 cells. A, shown is a schema of the construct for expression of the B56α-GFP fusion protein in HEK293 cells. The cDNA of either wild-type or mutated (S41A or S41D) B56α was subcloned in-frame

    Journal: The Journal of Biological Chemistry

    Article Title: Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41 *

    doi: 10.1074/jbc.M113.507996

    Figure Lengend Snippet: Expression of GFP-tagged B56α in HEK293 cells. A, shown is a schema of the construct for expression of the B56α-GFP fusion protein in HEK293 cells. The cDNA of either wild-type or mutated (S41A or S41D) B56α was subcloned in-frame

    Article Snippet: Cultured HEK293 cells (see above) were maintained on 24-well plates (Corning Glass) and transfected using TurboFectTM in vitro transfection reagent (Fermentas) according to manufacturer's protocol.

    Techniques: Expressing, Construct

    Assessment of mitoribosomal misreading in in organello translation Autoradiography of in organello mitochondrial translation products derived from HEK293 wild‐type cells in the presence of tobramycin, incorporation of 35 S‐Met and 35 S‐Cys in MT‐CO1 protein. Following in organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in organello translation in the presence of 1,000 μM tobramycin. MT‐CO1 bands were quantified and ratio of 35 S‐Cys/ 35 S‐Met calculated, and the 35 S‐Cys/ 35 S‐Met ratio in the absence of tobramycin was set as 1 ( n = 3), error bars indicate SEM. Ratio of 35 S‐Cys/ 35 S‐Met‐labeled MT‐CO1 and MT‐CO2 proteins synthesized in in organello translation and effect of tobramycin. Following in organello translation in the presence of tobramycin (1,000 μM, 2,000 μM), proteins were immunoprecipitated and analyzed by autoradiography. MT‐CO1 and MT‐CO2 bands were quantified, and ratio of 35 S‐Cys/ 35 S‐Met incorporation was calculated ( n = 3), error bars indicate SEM; * P

    Journal: EMBO Reports

    Article Title: Mutant MRPS5 affects mitoribosomal accuracy and confers stress‐related behavioral alterations

    doi: 10.15252/embr.201846193

    Figure Lengend Snippet: Assessment of mitoribosomal misreading in in organello translation Autoradiography of in organello mitochondrial translation products derived from HEK293 wild‐type cells in the presence of tobramycin, incorporation of 35 S‐Met and 35 S‐Cys in MT‐CO1 protein. Following in organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in organello translation in the presence of 1,000 μM tobramycin. MT‐CO1 bands were quantified and ratio of 35 S‐Cys/ 35 S‐Met calculated, and the 35 S‐Cys/ 35 S‐Met ratio in the absence of tobramycin was set as 1 ( n = 3), error bars indicate SEM. Ratio of 35 S‐Cys/ 35 S‐Met‐labeled MT‐CO1 and MT‐CO2 proteins synthesized in in organello translation and effect of tobramycin. Following in organello translation in the presence of tobramycin (1,000 μM, 2,000 μM), proteins were immunoprecipitated and analyzed by autoradiography. MT‐CO1 and MT‐CO2 bands were quantified, and ratio of 35 S‐Cys/ 35 S‐Met incorporation was calculated ( n = 3), error bars indicate SEM; * P

    Article Snippet: HEK293 cells were seeded on 24‐well plates (BD Falcon) at low density and incubated in DMEM with 10% FBS at 37°C.

    Techniques: Autoradiography, Derivative Assay, Immunoprecipitation, Labeling, Synthesized

    Analysis of HEK293 mutant MRPS5 V336Y and HEK293 MRPS5 wild‐type cells Generation time ( n = 8 clones, ± SD). Whole cell in vivo mitochondrial translation as determined by 35 S‐Met incorporation ( n = 4 clones, ± SEM). Autoradiography of in organello translation using 35 S‐Met labeling. Top five GO terms (cellular component) associated with genes upregulated in HEK293 mutant MRPS5 V336Y compared to HEK293 wild‐type MRPS5 . Corresponding GO terms associated with genes upregulated in mutant MT‐RNR1 A1555G lymphocytes (Hollis et al , 2015) were plotted for comparison. The bars represent the percentage of genes that map to a GO term and which are upregulated. Fisher's exact t ‐test, + P

    Journal: EMBO Reports

    Article Title: Mutant MRPS5 affects mitoribosomal accuracy and confers stress‐related behavioral alterations

    doi: 10.15252/embr.201846193

    Figure Lengend Snippet: Analysis of HEK293 mutant MRPS5 V336Y and HEK293 MRPS5 wild‐type cells Generation time ( n = 8 clones, ± SD). Whole cell in vivo mitochondrial translation as determined by 35 S‐Met incorporation ( n = 4 clones, ± SEM). Autoradiography of in organello translation using 35 S‐Met labeling. Top five GO terms (cellular component) associated with genes upregulated in HEK293 mutant MRPS5 V336Y compared to HEK293 wild‐type MRPS5 . Corresponding GO terms associated with genes upregulated in mutant MT‐RNR1 A1555G lymphocytes (Hollis et al , 2015) were plotted for comparison. The bars represent the percentage of genes that map to a GO term and which are upregulated. Fisher's exact t ‐test, + P

    Article Snippet: HEK293 cells were seeded on 24‐well plates (BD Falcon) at low density and incubated in DMEM with 10% FBS at 37°C.

    Techniques: Mutagenesis, In Vivo, Autoradiography, Labeling

    GP73 interacts and co-localizes with MAVS and TRAF6. ( A ) HEK293 cells (1×10 5 ) were co-transfected with GP73 expressing plasmid (0.1 μg) and ISRE or NF-κB reporters (0.05 μg) along with other plasmids as indicated (0.1 μg). Luciferase assays were performed 20 h after transfection. ( B ) HEK293 cells (2×10 6 ) were co-transfected with GP73 expressing plasmid (1 μg) and plasmids as indicated (3 μg) for 24 h. Cells were lysed and lysates were immunoprecipitated with anti-Flag or IgG. Immunoprecipitates and whole cell lysates (WCLs) were analyzed by WB with anti-HA or anti-Flag. ( C ) HEK293 cells (2×10 6 ) were co-transfected with HA- GP73-Myc (2 μg) and Flag- MAVS or Flag- TRAF6 (3 μg) for 24 h. Cells were lysed and lysates were immunoprecipitated with anti-Flag. Immunoprecipitates and WCLs were analyzed by WB with indicated antibodies. ( D ) HEK293 cells (2×10 7 ) were infected with SeV for indicated times. Immunoprecipitation and WB analysis were performed with antibodies against indicated proteins. ( E ) Huh7-MAVSR cells (5×10 7 ) were infected with HCV (MOI = 2) for the indicated days. Immunoprecipitation and WB analysis were performed with antibodies against indicated proteins. ( F ) HEK293 cells (5×10 5 ) were transfected with plasmids as indicated (2 μg) for 24 h. Cell lysates were subjected to GST pull-down assays with equal molar quantity of purified GST (10 μg) or recombinant GST-GP73 (20 μg) proteins. Immunoblots were performed with indicated antibodies. ( G ) HeLa cells (1×10 5 ) were co-transfected with HA-tagged GP73 plasmid (0.3 μg) and Flag-tagged MAVS or TRAF6 plasmids (0.7 μg) for 36 h. Cells were fixed and stained with a rabbit anti-HA monoclonal antibody (MAb) and a mouse anti-Flag MAb, followed by the FITC-conjugated goat anti-rabbit secondary antibody and a DyLight649-conjugated goat anti-mouse secondary antibody before confocal microscopy analysis. Nuclei were counterstained with DAPI. Scale bar, 10 μm. ( H ) HEK293 cells (1×10 7 ) were left un-treated or treated with SeV for 8 h and fractionated with differential centrifugation, the P5K and P50K fractions were subjected to WB with the indicated antibodies. Bar graphs represent means ± SD, ** P

    Journal: PLoS Pathogens

    Article Title: GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation

    doi: 10.1371/journal.ppat.1006321

    Figure Lengend Snippet: GP73 interacts and co-localizes with MAVS and TRAF6. ( A ) HEK293 cells (1×10 5 ) were co-transfected with GP73 expressing plasmid (0.1 μg) and ISRE or NF-κB reporters (0.05 μg) along with other plasmids as indicated (0.1 μg). Luciferase assays were performed 20 h after transfection. ( B ) HEK293 cells (2×10 6 ) were co-transfected with GP73 expressing plasmid (1 μg) and plasmids as indicated (3 μg) for 24 h. Cells were lysed and lysates were immunoprecipitated with anti-Flag or IgG. Immunoprecipitates and whole cell lysates (WCLs) were analyzed by WB with anti-HA or anti-Flag. ( C ) HEK293 cells (2×10 6 ) were co-transfected with HA- GP73-Myc (2 μg) and Flag- MAVS or Flag- TRAF6 (3 μg) for 24 h. Cells were lysed and lysates were immunoprecipitated with anti-Flag. Immunoprecipitates and WCLs were analyzed by WB with indicated antibodies. ( D ) HEK293 cells (2×10 7 ) were infected with SeV for indicated times. Immunoprecipitation and WB analysis were performed with antibodies against indicated proteins. ( E ) Huh7-MAVSR cells (5×10 7 ) were infected with HCV (MOI = 2) for the indicated days. Immunoprecipitation and WB analysis were performed with antibodies against indicated proteins. ( F ) HEK293 cells (5×10 5 ) were transfected with plasmids as indicated (2 μg) for 24 h. Cell lysates were subjected to GST pull-down assays with equal molar quantity of purified GST (10 μg) or recombinant GST-GP73 (20 μg) proteins. Immunoblots were performed with indicated antibodies. ( G ) HeLa cells (1×10 5 ) were co-transfected with HA-tagged GP73 plasmid (0.3 μg) and Flag-tagged MAVS or TRAF6 plasmids (0.7 μg) for 36 h. Cells were fixed and stained with a rabbit anti-HA monoclonal antibody (MAb) and a mouse anti-Flag MAb, followed by the FITC-conjugated goat anti-rabbit secondary antibody and a DyLight649-conjugated goat anti-mouse secondary antibody before confocal microscopy analysis. Nuclei were counterstained with DAPI. Scale bar, 10 μm. ( H ) HEK293 cells (1×10 7 ) were left un-treated or treated with SeV for 8 h and fractionated with differential centrifugation, the P5K and P50K fractions were subjected to WB with the indicated antibodies. Bar graphs represent means ± SD, ** P

    Article Snippet: Lentivirus were produced in HEK293 cells by co-transfection of pMD2.G (Addgene #12259), psPAX2 (Addgene #12260) and pLKO.1-shRNA plasmids (Ratio 1:3:4).

    Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Immunoprecipitation, Western Blot, Infection, Purification, Recombinant, Staining, Confocal Microscopy, Centrifugation

    TRAF-interacting-motifs are required for the interaction of MAVS with GP73. ( A ) Scheme of TRAF6 protein and its mutants (top panel). HEK293 cells (2×10 6 ) were co-transfected with GP73 (1 μg) and indicated plasmids (3 μg) for 24 h. Cell lysates were subjected to immunoprecipitation and WB with indicated antibodies. ( B ) Scheme of MAVS and its mutants. ( C , D , E and F ) Interactions of GP73 with MAVS and a series of truncations (C, D and E) or a series of TIM3 mutants (F). HEK293 cells (2×10 6 ) were co-transfected with GP73 plasmid (1 μg) and indicated plasmids (3 μg) for 24 h. Cell lysates were subjected to immunoprecipitation and WB with indicated antibodies.

    Journal: PLoS Pathogens

    Article Title: GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation

    doi: 10.1371/journal.ppat.1006321

    Figure Lengend Snippet: TRAF-interacting-motifs are required for the interaction of MAVS with GP73. ( A ) Scheme of TRAF6 protein and its mutants (top panel). HEK293 cells (2×10 6 ) were co-transfected with GP73 (1 μg) and indicated plasmids (3 μg) for 24 h. Cell lysates were subjected to immunoprecipitation and WB with indicated antibodies. ( B ) Scheme of MAVS and its mutants. ( C , D , E and F ) Interactions of GP73 with MAVS and a series of truncations (C, D and E) or a series of TIM3 mutants (F). HEK293 cells (2×10 6 ) were co-transfected with GP73 plasmid (1 μg) and indicated plasmids (3 μg) for 24 h. Cell lysates were subjected to immunoprecipitation and WB with indicated antibodies.

    Article Snippet: Lentivirus were produced in HEK293 cells by co-transfection of pMD2.G (Addgene #12259), psPAX2 (Addgene #12260) and pLKO.1-shRNA plasmids (Ratio 1:3:4).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation

    GP73 represses host innate immunity during viral infection. ( A ) HEK293 cells (1×10 5 ) were transfected with the IFN-β , ISRE or NF-κB reporters (0.1 μg) and GP73 expressing plasmid at indicated concentrations for 24 h, and infected with SeV for 10 h before luciferase reporter assays were performed. ( B ) HEK293 cells (1×10 5 ) were transfected with IFN-β or NF-κB reporter (0.05 μg) and GP73 -shRNAs expressing plasmids (1 μg) for 48 h, and infected with SeV for 10 h before reporter assays were performed. ( C , D ) GP73 -shRNAs transduced stable Huh7-MAVSR cells were transfected with in vitro transcribed JFH1 genomic RNA for 16 h. IFN-β , IL-6 and ISG56 mRNAs were quantified by RT-PCR (C) and p-IRF3 and p-IκBα were detected by WB (D). ( E , F ) GP73 -shRNAs transduced stable Huh7 ( E ) and THP-1 ( F ) cells were infected with SeV for 12 h. IFN-β , IFN-λl , IL-6 and TNF-α mNRAs were quantified by RT-PCR. Bar graphs represent means ± SD, * P

    Journal: PLoS Pathogens

    Article Title: GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation

    doi: 10.1371/journal.ppat.1006321

    Figure Lengend Snippet: GP73 represses host innate immunity during viral infection. ( A ) HEK293 cells (1×10 5 ) were transfected with the IFN-β , ISRE or NF-κB reporters (0.1 μg) and GP73 expressing plasmid at indicated concentrations for 24 h, and infected with SeV for 10 h before luciferase reporter assays were performed. ( B ) HEK293 cells (1×10 5 ) were transfected with IFN-β or NF-κB reporter (0.05 μg) and GP73 -shRNAs expressing plasmids (1 μg) for 48 h, and infected with SeV for 10 h before reporter assays were performed. ( C , D ) GP73 -shRNAs transduced stable Huh7-MAVSR cells were transfected with in vitro transcribed JFH1 genomic RNA for 16 h. IFN-β , IL-6 and ISG56 mRNAs were quantified by RT-PCR (C) and p-IRF3 and p-IκBα were detected by WB (D). ( E , F ) GP73 -shRNAs transduced stable Huh7 ( E ) and THP-1 ( F ) cells were infected with SeV for 12 h. IFN-β , IFN-λl , IL-6 and TNF-α mNRAs were quantified by RT-PCR. Bar graphs represent means ± SD, * P

    Article Snippet: Lentivirus were produced in HEK293 cells by co-transfection of pMD2.G (Addgene #12259), psPAX2 (Addgene #12260) and pLKO.1-shRNA plasmids (Ratio 1:3:4).

    Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Luciferase, In Vitro, Reverse Transcription Polymerase Chain Reaction, Western Blot

    GP73 facilitates proteasome-dependent degradation of MAVS and TRAF6. ( A ) HEK293 cells (2×10 5 ) were co-transfected with GP73 plasmids at 0, 0.125, 0.25 or 0.5 μg and β-actin plasmid (0.05 μg) along with MAVS or TRAF6 plasmids (0.5 μg) for 24 h. WCLs were subjected to WB with indicated antibodies. ( B ) HEK293 cells (5×10 5 ) were co-transfected with GP73 plasmids at 0, 0.5, 1, 1.5 or 2 μg for 24 h. Endogenous MAVS was determined by WB. ( C ) THP-1-GP73-RNAi cells were collected and endogenous MAVS, TRAF6, IRF3 and IκBα proteins were detected by WB. ( D ) HEK293 cells (5×10 5 ) were transfected with control or GP73 plasmids for 24 h, the mRNA levels of MAVS and TRAF6 were determined by RT-PCR (left panel). THP-1-GP73-RNAi cells were collected to determine the mRNA levels of MAVS and TRAF6 by RT-PCR (right panel). ( E ) Huh7-MAVSR cells were un-infected or infected with HCV (MOI = 2) for 4 days, CHX (cycloheximide, 40 μg/ml) were added for the indicated time points followed by WB analysis. ( F ) HEK293 cells (2×10 5 ) were transfected with GP73 plasmid (1 μg) for 20 h followed by treatment with DMSO, proteasome inhibitor MG132 (20 μM), autophagosome inhibitor 3-MA (1 mg/ml), lysosome inhibitor CQ (Chloroquine, 100 μM) or apoptosis inhibitor Z-FAD-FMK (50 μM) for 4 h. Endogenous MAVS was detected by WB. ( G ) HEK293 cells (2×10 5 ) were co-transfected with GP73 plasmid (0.1 μg) together with TRAF6 plasmid (0.2 μg) for 20 h followed by treatment with DMSO, proteasome inhibitor MG132 (20 μM) or BTZ (Bortezomib, 10 μM), lysosome inhibitor CQ (100 μM) or ER-to-Golgi transport inhibitor BFA (Brefeldin A, 20 μg/ml) for 4 h. WCLs were subjected to immunoblots with the indicated antibodies.

    Journal: PLoS Pathogens

    Article Title: GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation

    doi: 10.1371/journal.ppat.1006321

    Figure Lengend Snippet: GP73 facilitates proteasome-dependent degradation of MAVS and TRAF6. ( A ) HEK293 cells (2×10 5 ) were co-transfected with GP73 plasmids at 0, 0.125, 0.25 or 0.5 μg and β-actin plasmid (0.05 μg) along with MAVS or TRAF6 plasmids (0.5 μg) for 24 h. WCLs were subjected to WB with indicated antibodies. ( B ) HEK293 cells (5×10 5 ) were co-transfected with GP73 plasmids at 0, 0.5, 1, 1.5 or 2 μg for 24 h. Endogenous MAVS was determined by WB. ( C ) THP-1-GP73-RNAi cells were collected and endogenous MAVS, TRAF6, IRF3 and IκBα proteins were detected by WB. ( D ) HEK293 cells (5×10 5 ) were transfected with control or GP73 plasmids for 24 h, the mRNA levels of MAVS and TRAF6 were determined by RT-PCR (left panel). THP-1-GP73-RNAi cells were collected to determine the mRNA levels of MAVS and TRAF6 by RT-PCR (right panel). ( E ) Huh7-MAVSR cells were un-infected or infected with HCV (MOI = 2) for 4 days, CHX (cycloheximide, 40 μg/ml) were added for the indicated time points followed by WB analysis. ( F ) HEK293 cells (2×10 5 ) were transfected with GP73 plasmid (1 μg) for 20 h followed by treatment with DMSO, proteasome inhibitor MG132 (20 μM), autophagosome inhibitor 3-MA (1 mg/ml), lysosome inhibitor CQ (Chloroquine, 100 μM) or apoptosis inhibitor Z-FAD-FMK (50 μM) for 4 h. Endogenous MAVS was detected by WB. ( G ) HEK293 cells (2×10 5 ) were co-transfected with GP73 plasmid (0.1 μg) together with TRAF6 plasmid (0.2 μg) for 20 h followed by treatment with DMSO, proteasome inhibitor MG132 (20 μM) or BTZ (Bortezomib, 10 μM), lysosome inhibitor CQ (100 μM) or ER-to-Golgi transport inhibitor BFA (Brefeldin A, 20 μg/ml) for 4 h. WCLs were subjected to immunoblots with the indicated antibodies.

    Article Snippet: Lentivirus were produced in HEK293 cells by co-transfection of pMD2.G (Addgene #12259), psPAX2 (Addgene #12260) and pLKO.1-shRNA plasmids (Ratio 1:3:4).

    Techniques: Transfection, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Infection