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  • 99
    ATCC 293t cells 293t cells
    Reactivity of 2H2 to MED25 and common epitope. ( A ) Cross-reactivity of the 2H2 mAb against the common epitope. 2H2 mAb was generated using the common peptide (PPGAPKP) coupled on OVA. The cross-reactivity was detected by Western blot. 1 μg VP1, 5 μg OVA-P and 5 μg OVA were loaded on the gel. 2H2 mAb was used at a final concentration of 2 μg/mL. Specific bands are indicated by the red arrows. OVA, vector; OVA-P, vector with the common epitope peptide; ( B ) The lysate of <t>293T</t> cells transfected with MED25 expression vector was stained by anti-His, cells were transfected with the empty expression vector as a control. MED25 was probed by these antibodies in an independent Western blot experiment, and 10 5 cells lysate was loaded in each cell on gel. Specific band is indicated by the red arrow; ( C ) The lysate was stained by anti-MED25 commercial polyclonal antibody, and the other conditions were the same as panel B; ( D ) The lysate was stained by 2H2, and the other conditions were the same as panel B; ( E ) 293T cells transfected with MED25 expression plasmid and empty plasmid (control), respectively, were indirectly stained with 2H2 (FITC, green) and anti-MED25 (rhodamine, red) antibodies. The nuclei were stained with DAPI (blue). Merge 1: green + red. Merge 2: green + red + blue. The isotype antibody was included as the control to monitor specific staining. Images were obtained at a magnification of 400×.
    293t Cells 293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 293t cells 293t cells
    An N-terminal fragment corresponding to residues 175–244 of SOCS5 can directly bind JAK1. (A) SPR analysis of SOCS5 175–244 fragment binding to the JAK JH1 domain. Serially diluted JAK JH1 domains (62.5 nM–2 μM) were flowed over immobilised SOCS5 175–244 protein. Upper panels represent sensorgrams showing the kinetics of binding. Lower panels show steady-state analysis. (B) <t>293T</t> cells were transfected with the Stat6 reporter and increasing amounts of cDNA expressing Flag-tagged SOCS5 (3.13–100 ng) or SOCS5 lacking the conserved N-terminal fragment (9.5–300 ng; Δ175–244) and stimulated overnight with 10 ng/mL rhIL-4. Cells were lysed and induced luciferase activity measured and normalised according to Renilla activity. Data are expressed as arbitrary units and represent the mean of triplicates ± SD. Cell lysates were analyzed by Western blotting for Flag-tagged proteins (SOCS5 upper; Δ175–244 lower panel); images were generated from the same gel and exposure. (C) Recombinant SOCS5 JIR or SOCS3 was incubated with 20 nM JAK1 and GST-JAK2 activation peptide (substrate; GST-J) for 15 min in the presence of 2.5 mM Mg/ 32 P-γ-ATP at 37°C. Incorporation of 32 P was visualised by autoradiography (top panel) and protein input by SDS-PAGE and Coomassie staining (lower panel).
    293t Cells 293t Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 293t cells
    Direct binding measurements of CD4-Ig and mabs follows neutralization sensitivity of Env+ pseudoviruses. LN8 wt, 375W and 380P Envs were expressed on <t>293T</t> cells before measuring binding of CD4-Ig and mabs using flow cytometry. Boxed values in the right hand, top corner of each flow profile represents the neutralization titer for each reagent and shows that binding closely followed neutralization sensitivity.
    293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29983 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Cell Genesys 293t cells
    Socs-1 inhibits kinase activity of TEL-JAK2. (A) TEL-JAK2 in vitro kinase assay. <t>293T</t> cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc–Socs-1 were starved of FBS for 4 h. Extracts from these cells were immunoprecipitated (IP) with a TEL antibody and used for in vitro kinase assays using GST-C′Gab2 as a substrate. Products were separated by SDS-PAGE and visualized by autoradiography (top). Parallel TEL Western blotting confirmed equal immunoprecipitation of the protein (bottom). (B) 4G10 Western blot. 293T cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc-Socs-1 were starved of FBS for 4 h. TEL-JAK2 was immunoprecipitated with the JAK2 antibody and blotted with 4G10 (top) or JAK2 antibody (bottom). The whole-cell lysates were blotted with a Myc antibody to confirm expression of Socs-1 and a TEL antibody to confirm equal expression of TEL-JAK2 (data not shown). (C) Phosphoamino acid analysis of C′Gab2. Phosphorylated GST-C′Gab2 substrate from panel was hydrolyzed to single amino acids and separated on a thin-layer chromatography plate (left). The plate was placed on a phosphorimager, and the ratio of phosphorylated amino acids was used to generate the graph (right). Open bars represent TEL-JAK2 samples, and solid bars represent Socs-1 and TEL-JAK2 samples. (D) Phosphoamino acid analysis of autophosphorylated TEL-JAK2. Analysis of the autophosphorylated TEL-JAK2 was performed as for panel C. Open bars represent TEL-JAK2 samples, and solid bars represent Socs-1 and TEL-JAK2 samples.
    293t Cells, supplied by Cell Genesys, used in various techniques. Bioz Stars score: 87/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Mirus Bio 293t cells
    Exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles. (A) BE-KO1 cells infected with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA1 (A1), ApoA2 (A2), ApoC1 (C1), ApoC2 (C2), ApoC3 (C3) and ApoH (H) and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. (B) ApoA1, ApoA2, ApoC1, ApoC2, ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with HCVcc at an MOI of 1. Expression of the apolipoproteins was determined by immunoblot analysis (upper), and infectious titers in the culture supernatants were determined at 72 h post-infection by focus-forming assay (lower). (C) Extracellular and intracellular HCV RNA in BE-KO1 cells expressing apolipoproteins and infected with HCVcc were determined at 72 h post-infection by qRT-PCR. (D) Specific infectivity was calculated as extracellular infectious titers/extracellular HCV RNA copies in BE-KO1 cells expressing apolipoproteins at 72 h post-infection. (E) <t>293T</t> cells stably expressing CLDN1 and miR-122 (293T-CLDN/miR-122 cells) were infected with the lentiviral vectors, and the expressions of the apolipoproteins were determined by immunoblot analysis (upper). These cells were infected with HCVcc at an MOI of 1, and infectious titers in the supernatants were determined at 72 h post-infection by focus-forming assay (lower). In all cases, asterisks indicate significant differences (*, P
    293t Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 99/100, based on 2560 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hek 293 cells
    Albumin led to the reduction of intracellular superoxide anion levels induced by hydrogen peroxide in human kidney cells. (A) Representative image of intracellular superoxide anion levels as detected by dihydroethidium (DHE) staining in human embryonic kidney (HEK) 293FT cells after 6-h exposure to hydrogen peroxide (500 μM) and pretreatment with phosphate buffered saline (PBS), human serum albumin (HSA, 3.0 g/dL) or γ-globulin (3.0 g/dL). As a control (CTR), we used cells that were not exposed to hydrogen peroxide. (B) The DHE fluorescent intensity per cell was quantified. <t>HEK</t> 293 FT cells were exposed to 500 μM hydrogen peroxide for 6 h with PBS, HSA, or γ-globulin. As a CTR, we used cells that were not exposed to hydrogen peroxide. Fluorescent intensities per cell were measured using ImageJ software. Data are presented as the mean ± standard error. (n = 8 imaged fields for each condition). * P
    Hek 293 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche 293t cells
    Lentiviral-mediated gene delivery to the mouse small intestine . a Schematic of virus production in <t>293T</t> cells and mouse injection. Mice were injected at postnatal day 1 and sacrificed for analysis at later time points. b Green fluorescent protein (GFP) gene expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in the gastrointestinal tract and other organs. GFP expression is normalized to glyceraldehyde phosphate dehydrogenase expression and presented relative to levels in the stomach. Graph displays triplicate samples from representative animals. Data displayed as mean ± std. c GFP expression in isolated intestinal crypt and villus epithelium and mesenchymal cells by qRT-PCR. Graph displays triplicate samples from representative animal. Data displayed as mean ± std. d Hematoxylin and eosin stained normal intestinal crypt-villus unit. Solid line indicates apical epithelial border; dashed line indicates epithelial-mesenchymal border. e–g Co-labeling of DsRed-injected intestine with antibodies to GFP ( green ) and DsRed ( red ). Yellow box in e and f is enlarged in g. Arrowheads indicate dual expressing mesenchymal cells. Dashed line indicates epithelial-mesenchymal border. Solid white line in g marks the apical border. Bar = 25 μm.
    293t Cells, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 7955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Genechem 293t cells
    Lentiviral-mediated gene delivery to the mouse small intestine . a Schematic of virus production in <t>293T</t> cells and mouse injection. Mice were injected at postnatal day 1 and sacrificed for analysis at later time points. b Green fluorescent protein (GFP) gene expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in the gastrointestinal tract and other organs. GFP expression is normalized to glyceraldehyde phosphate dehydrogenase expression and presented relative to levels in the stomach. Graph displays triplicate samples from representative animals. Data displayed as mean ± std. c GFP expression in isolated intestinal crypt and villus epithelium and mesenchymal cells by qRT-PCR. Graph displays triplicate samples from representative animal. Data displayed as mean ± std. d Hematoxylin and eosin stained normal intestinal crypt-villus unit. Solid line indicates apical epithelial border; dashed line indicates epithelial-mesenchymal border. e–g Co-labeling of DsRed-injected intestine with antibodies to GFP ( green ) and DsRed ( red ). Yellow box in e and f is enlarged in g. Arrowheads indicate dual expressing mesenchymal cells. Dashed line indicates epithelial-mesenchymal border. Solid white line in g marks the apical border. Bar = 25 μm.
    293t Cells, supplied by Genechem, used in various techniques. Bioz Stars score: 97/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Shanghai Genechem 293t cells
    Overexpression of p53 by constructing lentivirus. The protein CDS region of murine p53 gene was inserted into the lentiviral expression vector pLVX-puro. Lentivirus encoding p53 (pLVX-p53) and control virus (pLVX) were packaged in <t>293T</t> cells and used to infect MC3T3-E1 cells. We evaluated (A) mRNA levels of p53 with qPCR and (B) protein levels of p53 with western blotting, at 48 h after viral infection. ***P
    293t Cells, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 94/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    System Biosciences Inc 293t cells
    LPS promotes RIP140 degradation through SOCS1-Rbx1 E3 ligase-mediated K48 polyubiquitination. (a) Immunoblot analysis of RIP140 and polyubiquitinated RIP140 in Raw264.7 macrophages stimulated for 6 h with indicated treatments. (b) Immunoblot analysis of RIP140, Rbx1 and actin in primary peritoneal macrophages stimulated for 24 h with a control treatment or LPS after transfection with control or Rbx1 siRNAs. (c) Immunoblot analysis of RIP140, SOCS1 and actin in primary peritoneal macrophages stimulated for 24 h with a control treatment or LPS after trasfection with control or SOCS1 siRNAs. (d, e, f) In vitro ubiqtuination analysis of RIP140 in <t>293T</t> cells. 293T cells were transfected with expression plasmids as indicated and treated with 10 μM MG132 for 6 h before lysis. (d) Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-Rbx1. (e) Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-SOCS1. (f) 293T cells were transfected with Flag-tagged RIP140 and HA-tagged Ub in conjunction with HA-tagged SOCS1 wild type (Wt) or HA-tagged SOCS1-ΔSH (ΔSH). Cells were treated with 10 μM MG132 for 6 h before lysis. Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-SOCS1. All immunoblots were performed at least twice.
    293t Cells, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 96/100, based on 376 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    GenePharma Company 293t cells
    Vascular endothelial growth factor C (VEGFC) is a novel target of miR-27b in colorectal cancer (CRC). (A) VEGFC is predicted as a novel target of miR-27b. (B) <t>293T</t> cells were co-transfected with empty pmirGLO Dual-Luciferase reporter plasmids or VEGFC 3′UTR firefly luciferase reporter plasmids and pRL-TK-luciferase plasmids, together with miR-27b mimics or anti-miR-27b. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase. (C) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and expression of VEGFC was detected by western blotting. (D) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and VEGFC in culture medium was detected by ELISA. (E) VEGFC protein in xenografts from negative control (NC) and miR-27b mimics was detected by western blotting. Error bars represent the means ± SEM, * P
    293t Cells, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 98/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Orbigen 293t cells
    Vascular endothelial growth factor C (VEGFC) is a novel target of miR-27b in colorectal cancer (CRC). (A) VEGFC is predicted as a novel target of miR-27b. (B) <t>293T</t> cells were co-transfected with empty pmirGLO Dual-Luciferase reporter plasmids or VEGFC 3′UTR firefly luciferase reporter plasmids and pRL-TK-luciferase plasmids, together with miR-27b mimics or anti-miR-27b. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase. (C) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and expression of VEGFC was detected by western blotting. (D) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and VEGFC in culture medium was detected by ELISA. (E) VEGFC protein in xenografts from negative control (NC) and miR-27b mimics was detected by western blotting. Error bars represent the means ± SEM, * P
    293t Cells, supplied by Orbigen, used in various techniques. Bioz Stars score: 87/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sangon Biotech 293t cells
    Promoter analysis of the sequence upstream of the human hnRNP K gene. A. Promoter luciferase reporter analysis in <t>293T,</t> MCF-7 and HK2 cells. The relative promoter activities are represented as the fold increase versus expression from the activities of renilla plasmid. Values represent the mean ± SD of three independent experiments. T -test was used for statistical analysis. (a) P
    293t Cells, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 97/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza 293t cells
    T cell multiepitopic B (TMEP-B) design, construction of pcDNA-TMEP-B plasmid, and TMEP-B expression analysis by Western blot. ( A ) Scheme of TMEP-B protein. ( B ) Map of the plasmid pcDNA-TMEP-B. ( C ) Expression of TMEP-B construct by Western blot. The <t>293T</t> cells were mock-infected or infected with 5 pfu/cell of Western Reserve (WR) or vaccinia virus (VACV) that expresses the T7 RNA polymerase (VT7) viruses, and transfected 1 h later with 5 μg of pcDNA-TMEP-B or pMax-GFP. At 6 h post-infection, cells were harvested and lysed in Laemmli buffer with mercapoethanol and cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody to evaluate TMEP-B expression.
    293t Cells, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    DSMZ hek 293 cells
    Structure of sgp130-E10 and its interaction with Hyper-IL-6. A , schematic overview of sgp130-E10 in complex with IL-6/IL-6R. B , immunoprecipitation with conditioned medium from <t>HEK-293</t> cell cultures transiently transfected with plasmids coding for sgp130-E10Myc-His
    Hek 293 Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa hek 293 cells
    Current-voltage relationship of SARS-CoV E protein and inhibition by HMA. (A) Example traces of current flowing through cells expressing SARS-CoV E protein, vector alone-transfected cells and untransfected <t>HEK-293</t> cells. The cells were held at 0 mV and stepped to various potentials from −100 to 70 mV (in steps of 10 mV). (B) Whole-cell I–V curve in which peak current amplitudes were plotted against test potentials. Notice the significant large inward and outward currents recorded from SARS-CoV E protein, in contrast to vector alone and untransfected HEK-293 cell controls (*, two-tail unpaired T test, p
    Hek 293 Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 458 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cyagen Biosciences 293t cells
    Current-voltage relationship of SARS-CoV E protein and inhibition by HMA. (A) Example traces of current flowing through cells expressing SARS-CoV E protein, vector alone-transfected cells and untransfected <t>HEK-293</t> cells. The cells were held at 0 mV and stepped to various potentials from −100 to 70 mV (in steps of 10 mV). (B) Whole-cell I–V curve in which peak current amplitudes were plotted against test potentials. Notice the significant large inward and outward currents recorded from SARS-CoV E protein, in contrast to vector alone and untransfected HEK-293 cell controls (*, two-tail unpaired T test, p
    293t Cells, supplied by Cyagen Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 293t cells
    Effect of LR1-C1 mutations on affinity for selected bNAbs. Virus stocks were obtained from transfected <t>293T</t> cells. Increase in Ab binding was determined by comparison of the virus captured by different monoclonal antibodies and quantified by p24 when similar amounts of virus (AC10_29 in red and filled circles and LR1-C1 in blue and filled squares) were used as input in a Virion Capture Assay (VCA) with the Abs 4E10 (MPER), 2F5 (MPER), 447-52D (V3), VRC01 (CD4bs), 2G12 (N332 V3 glycan patch), b12 (CD4bs), PG16 (quaternary; V1-V1 glycan apex), PGT151 (quaternary; gp120-gp41 integrase) and 5F3 (gp41). Similar amounts of virus suspension with no mAb were used as controls for both viruses (AC10_29 in grey and filled circles and LR1-C1 in grey and filled squares). Statistical analysis was conducted by R using One-way NOVA followed by Newman Keuls post hoc test at each input concentration (***p
    293t Cells, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen hek 293 cells
    Higher expression of specific genes in cells expressing S547A mutated p65. qRT PCR analysis of IL8 ( A ), A20 ( B ), Sele ( C ), VCAM1 ( D ), CXCL1 ( E ) and CD83 ( F ) mRNA level after 4 and 8 hours of etoposide treatment in <t>HEK-293</t> cells expressing either p65 wt or p65 S547A .
    Hek 293 Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 272 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson 293t cells
    Targeted transduction and delivery of PSCA antigen gene into dendritic cells (DCs) by DCLV-PSCA. (A) <t>293T</t> cells were transfected transiently with plasmids FUW-Null (mock control, blue line) or FUW-PSCA (red line). Two days later, cells were collected and stained for PSCA expression analyzed by flow cytometry. 293T cells stained with the isotype antibody were included as a control (grey shade area). (B) 293T cells were transfected transiently with plasmids FUW-PSCA, SVGmu, and other necessary lentiviral packaging plasmids to produce DCLV-PSCA vectors. Fresh virus supernatant was used to transduce 293T cells (blue line) or 293T.hDC-SIGN cells (red line) with MOI = 10. PSCA expression was analyzed by flow cytometry 3 days post-transduction. (C) Bone marrow-derived DCs were transduced with a mock vector DC-LV-Null or DC-LV-PSCA vector. Five days later, CD11c and PSCA expression were assessed by flow cytometric analysis. All experiments were repeated three times and the representative data is shown.
    293t Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 957 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam 293t cells
    Fluorescent recombineering reporter. A) Lentiviral vector pDual-eGFP Y203 was used to deliver a Yellow florescent protein variant of eGFP to serve as the recombineering target transgene. B) Fluorescence excitation and emission spectra of purified eGFP and eGFP Y203 proteins. C-F) Flow cytometry was used to quantify <t>293T-Green</t> recombinant and 293T-Yellow parental phenotypes. C) Untransduced cells were quantified in the R1 gate, D) pDual-eGFP transduced Green cells were quantified in the P2 gate and E) pDual-eGFP Y203 transduced Yellow cells were quantified in the P3 gate. F) Example results from one recombination experiment using Yellow cells as a target and an oligo that codes for Green fluorescence.
    293t Cells, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biosettia 293t cells
    hsa-miR-29a-3p inhibited luciferase reporter gene expression controlled by the 3′-UTRs of ALDH5A1 or SLC22A7 (A) Free energy analyses for the interactions between hsa-miR-29a-3p and the targeting sequences or site-mutants present the in ALDH5A1 3′-UTR, or SLC22A7 3′-UTR. Δ G , free energy; the underlined letter, the mutated base. (B) ALDH5A1-CU and ALDH5A1-Mut plasmids or (C) SLC22A7-CU and SLC22A7-Mut plasmids were transiently transfected into <t>293T</t> and HepG2 cells, together with 50 nmol/L hsa-miR-29a-3p mimic or miRNA negative control. Cells were harvested 48 h after transfection. Three independent experiments, each in triplicate, were performed, and fold changes of luciferase activity were calculated by defining the activity of ALDH5A1-CU plasmid, or SLC22A7-CU, together with miRNA negative control as unity. ** P
    293t Cells, supplied by Biosettia, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa lentix 293t parental 293t cells
    Quantitative analysis of TGF-β pathway activation by aqueous humor of patients and controls. Box and whisker plot showing that the aqueous humor of naïve nAMD patients induces less TGF-β pathway activation in <t>Lenti-X</t> <t>293T</t> cells. Cells were co-transfected with the plasmids pNL[NlucP/SBE], expressing NanoLuc luciferase under the control of three SMAD3 binding elements, and pGL4.54[luc2/TK], expressing the Firefly luciferase luc2 (used as transfection normalizer) under the control of the constitutive HSV-TK promoter. Transfected cells were treated with 10 μl of aqueous humor of the 20 nAMD patients, naïve or treated (once or twice, Treat.1 and Treat.2, respectively), or of the 20 control samples (Cntr.) described in Table 1 . Data are presented as the ratio between NanoLuc and luc2 luciferase activity, expressed as Relative Light Units (RLU) (number of replicates for each sample = 3). Asterisks indicate significant differences (**P
    Lentix 293t Parental 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam 293t cell lysates
    Quantitative analysis of TGF-β pathway activation by aqueous humor of patients and controls. Box and whisker plot showing that the aqueous humor of naïve nAMD patients induces less TGF-β pathway activation in <t>Lenti-X</t> <t>293T</t> cells. Cells were co-transfected with the plasmids pNL[NlucP/SBE], expressing NanoLuc luciferase under the control of three SMAD3 binding elements, and pGL4.54[luc2/TK], expressing the Firefly luciferase luc2 (used as transfection normalizer) under the control of the constitutive HSV-TK promoter. Transfected cells were treated with 10 μl of aqueous humor of the 20 nAMD patients, naïve or treated (once or twice, Treat.1 and Treat.2, respectively), or of the 20 control samples (Cntr.) described in Table 1 . Data are presented as the ratio between NanoLuc and luc2 luciferase activity, expressed as Relative Light Units (RLU) (number of replicates for each sample = 3). Asterisks indicate significant differences (**P
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    ATCC hek 293 cells
    DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) <t>HEK-293</t> cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
    Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tla 293t cells
    DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) <t>HEK-293</t> cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
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    TaKaRa ltx 293t cells
    DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) <t>HEK-293</t> cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
    Ltx 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa ecopack 293t cells
    DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) <t>HEK-293</t> cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
    Ecopack 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa aavpro 293t cells
    DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) <t>HEK-293</t> cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
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    Roche 293t cells 293t cells
    Human Jurkat T cells lentivirally transduced with Runx1.d190 show increased transcription of c-Myc . (A) Schematic of the structure of Runx1 and Runx1.d190. (B) <t>293T.</t> cells were transfected with empty pEGFP-N1 vector (EGFPonly, left column) as a control for cytoplasmic staining, pEGFP-N1 vector containing full-length Runx1 fused in-frame to EGFP (Runx1FL, middle column) or Runx1.d190 fused in-frame to EGFP (Runx1.d190, right column). The nuclear DNA was visualized by staining with Hoescht 33342 (Nuclear, top row). Nuclear (top row) and EGFP (middle row) fluorescence are shown in isolation and merged (Merged, bottom row). (C) Relative differences in transcription between Jurkat T cells lentivirally transduced with control empty vector or vector encoding Runx1.d190 as determined by microarray analysis are shown. A complete listing of genes whose transcription is affected by Runx1.d190 in Jurkat T cells is located at http://www.ncbi.nlm.nih.gov/geo/ . (D) ChIP analysis. Chromatin was prepared from Jurkat T cells lentivirally transduced with Runx1.d190 and immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out using primer sets amplifying Runx1-binding sites at -0.83 (i), -7.9 (ii) and -8.9 kb (iii) upstream of the human c-Myc transcriptional start site. N =3.
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    Image Search Results


    Reactivity of 2H2 to MED25 and common epitope. ( A ) Cross-reactivity of the 2H2 mAb against the common epitope. 2H2 mAb was generated using the common peptide (PPGAPKP) coupled on OVA. The cross-reactivity was detected by Western blot. 1 μg VP1, 5 μg OVA-P and 5 μg OVA were loaded on the gel. 2H2 mAb was used at a final concentration of 2 μg/mL. Specific bands are indicated by the red arrows. OVA, vector; OVA-P, vector with the common epitope peptide; ( B ) The lysate of 293T cells transfected with MED25 expression vector was stained by anti-His, cells were transfected with the empty expression vector as a control. MED25 was probed by these antibodies in an independent Western blot experiment, and 10 5 cells lysate was loaded in each cell on gel. Specific band is indicated by the red arrow; ( C ) The lysate was stained by anti-MED25 commercial polyclonal antibody, and the other conditions were the same as panel B; ( D ) The lysate was stained by 2H2, and the other conditions were the same as panel B; ( E ) 293T cells transfected with MED25 expression plasmid and empty plasmid (control), respectively, were indirectly stained with 2H2 (FITC, green) and anti-MED25 (rhodamine, red) antibodies. The nuclei were stained with DAPI (blue). Merge 1: green + red. Merge 2: green + red + blue. The isotype antibody was included as the control to monitor specific staining. Images were obtained at a magnification of 400×.

    Journal: Viruses

    Article Title: Identification of a Common Epitope between Enterovirus 71 and Human MED25 Proteins Which May Explain Virus-Associated Neurological Disease

    doi: 10.3390/v7041558

    Figure Lengend Snippet: Reactivity of 2H2 to MED25 and common epitope. ( A ) Cross-reactivity of the 2H2 mAb against the common epitope. 2H2 mAb was generated using the common peptide (PPGAPKP) coupled on OVA. The cross-reactivity was detected by Western blot. 1 μg VP1, 5 μg OVA-P and 5 μg OVA were loaded on the gel. 2H2 mAb was used at a final concentration of 2 μg/mL. Specific bands are indicated by the red arrows. OVA, vector; OVA-P, vector with the common epitope peptide; ( B ) The lysate of 293T cells transfected with MED25 expression vector was stained by anti-His, cells were transfected with the empty expression vector as a control. MED25 was probed by these antibodies in an independent Western blot experiment, and 10 5 cells lysate was loaded in each cell on gel. Specific band is indicated by the red arrow; ( C ) The lysate was stained by anti-MED25 commercial polyclonal antibody, and the other conditions were the same as panel B; ( D ) The lysate was stained by 2H2, and the other conditions were the same as panel B; ( E ) 293T cells transfected with MED25 expression plasmid and empty plasmid (control), respectively, were indirectly stained with 2H2 (FITC, green) and anti-MED25 (rhodamine, red) antibodies. The nuclei were stained with DAPI (blue). Merge 1: green + red. Merge 2: green + red + blue. The isotype antibody was included as the control to monitor specific staining. Images were obtained at a magnification of 400×.

    Article Snippet: Expression of MED25 in 293T Cells 293T cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle medium (DMEM) (Sigma, Saint Louis, MO, USA) supplemented with 10% Fetal bovine serum (FBS) (10% FBS-DMEM).

    Techniques: Generated, Western Blot, Concentration Assay, Plasmid Preparation, Transfection, Expressing, Staining

    GEP100 associates with Her2 to induce Arf6 activation. ( A ) Co-precipitation of Her2-EGFP with HA-GEP100, expressed in 293T cells, and analysed by anti-GEP100 immunoprecipitation (IP) coupled with anti-GFP immunoblots (IB). Anti-GEP100 immunoprecipitants were also blotted by anti-phospho Her2 and an anti-HA antibody. Immunoprecipitation for the expression of Her2-EGFP without HA-GEP100 was included as a control (vector). Immunoprecipitation using non-immune serum was also included as a control (NC). ( B ) In vitro co-precipitation of Her2-EGFP (pEGFP-Her2) expressed in cells with the two indicated GST-tagged PH domains (GST-GEP100-PH/GST-ARNO-PH) or GST alone, analysed by glutathione-beads pulldown and anti-GFP immunoblots. GST-fusion proteins were visualized by Ponceau S. In A and B , cells were cultured with 10% FCS (Se), in the absence of serum for 24 h (St), or stimulated with 10 ng/ml EGF for 10 min after serum starvation for 24 h (E), prior to lysis. EGFP alone was included as a control (pEGFP). ( C ) Co-precipitation of HA-GEP100 with wild type Her2-EGFP (WT) or its mutants (YF1; 1139F, YF2; 1196F, YF3; 1221/1222F, YF4; 1248F, YF1/2; 1139/1196F, YF3/4; 1221/1222/1248F), analysed by anti-GEP100 immunoprecipitation and anti-GFP immunoblots. ( D ) Arf6-myc activities in cells expressing HA-GEP100 and Her2-EGFP or their mutants, measured by GST-GGA pulldown and anti-myc immunoblots. +, wild type; YF, Her2 1139/1196F mutant; Del, Sec7-deleted GEP100. EGFP alone was included as a control (G). In A – D , immunoblots of total cell lysates (10 µg) are also shown (Total).

    Journal: PLoS ONE

    Article Title: Engagement of Overexpressed Her2 with GEP100 Induces Autonomous Invasive Activities and Provides a Biomarker for Metastases of Lung Adenocarcinoma

    doi: 10.1371/journal.pone.0025301

    Figure Lengend Snippet: GEP100 associates with Her2 to induce Arf6 activation. ( A ) Co-precipitation of Her2-EGFP with HA-GEP100, expressed in 293T cells, and analysed by anti-GEP100 immunoprecipitation (IP) coupled with anti-GFP immunoblots (IB). Anti-GEP100 immunoprecipitants were also blotted by anti-phospho Her2 and an anti-HA antibody. Immunoprecipitation for the expression of Her2-EGFP without HA-GEP100 was included as a control (vector). Immunoprecipitation using non-immune serum was also included as a control (NC). ( B ) In vitro co-precipitation of Her2-EGFP (pEGFP-Her2) expressed in cells with the two indicated GST-tagged PH domains (GST-GEP100-PH/GST-ARNO-PH) or GST alone, analysed by glutathione-beads pulldown and anti-GFP immunoblots. GST-fusion proteins were visualized by Ponceau S. In A and B , cells were cultured with 10% FCS (Se), in the absence of serum for 24 h (St), or stimulated with 10 ng/ml EGF for 10 min after serum starvation for 24 h (E), prior to lysis. EGFP alone was included as a control (pEGFP). ( C ) Co-precipitation of HA-GEP100 with wild type Her2-EGFP (WT) or its mutants (YF1; 1139F, YF2; 1196F, YF3; 1221/1222F, YF4; 1248F, YF1/2; 1139/1196F, YF3/4; 1221/1222/1248F), analysed by anti-GEP100 immunoprecipitation and anti-GFP immunoblots. ( D ) Arf6-myc activities in cells expressing HA-GEP100 and Her2-EGFP or their mutants, measured by GST-GGA pulldown and anti-myc immunoblots. +, wild type; YF, Her2 1139/1196F mutant; Del, Sec7-deleted GEP100. EGFP alone was included as a control (G). In A – D , immunoblots of total cell lysates (10 µg) are also shown (Total).

    Article Snippet: Cells 293T cells obtained from American Type Culture Collection were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS (Hyclone).

    Techniques: Activation Assay, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, In Vitro, Cell Culture, Lysis, Mutagenesis

    Replication of ROD10 and ROD14 Env viruses. a Viruses containing either the wild-type ROD10 Env [ROD10(WT Env)] or the mutant ROD14 Env protein that does not counteract tetherin [ROD10(14 Env)] were produced in 293T cells and 3 μg p27 equivalent of supernatants were used to infect 5 × 10 6 JLTRG cells. A fraction of the cells were analyzed by flow cytometry at day 4, then every 3 days, for GFP expression. b Virus from day 25 in the initial infections was transferred to fresh JLTRG cells and the cells monitored by flow cytometry. Infections were stopped at day 12, when it was observed that all viruses were replicating with wild-type kinetics.

    Journal: Retrovirology

    Article Title: Determinants in HIV-2 Env and tetherin required for functional interaction

    doi: 10.1186/s12977-015-0194-0

    Figure Lengend Snippet: Replication of ROD10 and ROD14 Env viruses. a Viruses containing either the wild-type ROD10 Env [ROD10(WT Env)] or the mutant ROD14 Env protein that does not counteract tetherin [ROD10(14 Env)] were produced in 293T cells and 3 μg p27 equivalent of supernatants were used to infect 5 × 10 6 JLTRG cells. A fraction of the cells were analyzed by flow cytometry at day 4, then every 3 days, for GFP expression. b Virus from day 25 in the initial infections was transferred to fresh JLTRG cells and the cells monitored by flow cytometry. Infections were stopped at day 12, when it was observed that all viruses were replicating with wild-type kinetics.

    Article Snippet: Cell lines 293T cells were obtained from the American Type Culture Collection; 293A cells were obtained from Qbiogene/MP Biomedicals (Irvine, CA, USA) and JLTRG cells [ ] were obtained from the AIDS Research, Reference, and Reagent Program (ARRRP).

    Techniques: Mutagenesis, Produced, Flow Cytometry, Cytometry, Expressing

    Deubiquitination of interferon regulatory factor 4 (IRF4) by ubiquitin specific peptidase 4 (USP4). USP4 can mediate deubiquitination of IRF4 on K48 and K63 sequences (amino acid residue sequences). 293T cells were cotransfected with Myc-IRF4, Ha-USP4, His-ubiquitin or His-ubiquitin mutant K63 only and K48 only. Cells were collected after 48 h; MG132 (20 µ M/ml) was used for processing and Ni-NTA was used for purified precipitation, and immunoblotting was conducted with monoclonal antibody of anti-Ha and anti-Flag.

    Journal: International Journal of Molecular Medicine

    Article Title: Ubiquitin specific peptidase 4 stabilizes interferon regulatory factor protein and promotes its function to facilitate interleukin-4 expression in T helper type 2 cells

    doi: 10.3892/ijmm.2017.3087

    Figure Lengend Snippet: Deubiquitination of interferon regulatory factor 4 (IRF4) by ubiquitin specific peptidase 4 (USP4). USP4 can mediate deubiquitination of IRF4 on K48 and K63 sequences (amino acid residue sequences). 293T cells were cotransfected with Myc-IRF4, Ha-USP4, His-ubiquitin or His-ubiquitin mutant K63 only and K48 only. Cells were collected after 48 h; MG132 (20 µ M/ml) was used for processing and Ni-NTA was used for purified precipitation, and immunoblotting was conducted with monoclonal antibody of anti-Ha and anti-Flag.

    Article Snippet: Cell culture 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT, USA) containing 10% FBS.

    Techniques: Mutagenesis, Purification

    Effect of ubiquitin specific peptidase 4 (USP4) on stabilization of interferon regulatory factor 4 (IRF4). IRF4 protein expression level was influenced by USP4 in a dose-dependent manner. Equivalent dose of Myc-IRF4 and different doses of Ha-USP4 were cotransfected into 293T cells. After 48 h, the cells were retrieved and the expression levels of USP4 and IRF4 were detected by immunoblotting.

    Journal: International Journal of Molecular Medicine

    Article Title: Ubiquitin specific peptidase 4 stabilizes interferon regulatory factor protein and promotes its function to facilitate interleukin-4 expression in T helper type 2 cells

    doi: 10.3892/ijmm.2017.3087

    Figure Lengend Snippet: Effect of ubiquitin specific peptidase 4 (USP4) on stabilization of interferon regulatory factor 4 (IRF4). IRF4 protein expression level was influenced by USP4 in a dose-dependent manner. Equivalent dose of Myc-IRF4 and different doses of Ha-USP4 were cotransfected into 293T cells. After 48 h, the cells were retrieved and the expression levels of USP4 and IRF4 were detected by immunoblotting.

    Article Snippet: Cell culture 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT, USA) containing 10% FBS.

    Techniques: Expressing

    Interaction between ubiquitin specific peptidase 4 (USP4) and interferon regulatory factor 4 (IRF4). (A) IRF4 was co-immunoprecipitated by the anti-Ha antibody when Ha-USP4 and Myc-IRF4 were coexpressed. (B) IRF4 was co-immunoprecipitated by the anti-Ha antibody when Ha-USP4 and Myc-IRF4 were coexpressed, and USP4 was co-immunoprecipitated by the anti-Myc antibody when Ha-USP4 and Myc-IRF4 were coexpressed. (C) IRF4 was co-immunoprecipitated by the anti-USP4 antibody, indicating that USP4 interacts with IRF4 in Th2 cells. Flag-IRF4 and Ha-USP4 were co-transfected into 293T cells. After 48 h, the cells were retrieved, and 1 µ g of anti-Ha or anti-Flag monoclonal antibody precipitate was added and immunoblotting was conducted in A and B. Th2 cells were amplified to 1×10 7 , and co-immunoprecipitated with 1 µ g of normal IgG or anti-USP4, then immunoblotting assay was conducted in C.

    Journal: International Journal of Molecular Medicine

    Article Title: Ubiquitin specific peptidase 4 stabilizes interferon regulatory factor protein and promotes its function to facilitate interleukin-4 expression in T helper type 2 cells

    doi: 10.3892/ijmm.2017.3087

    Figure Lengend Snippet: Interaction between ubiquitin specific peptidase 4 (USP4) and interferon regulatory factor 4 (IRF4). (A) IRF4 was co-immunoprecipitated by the anti-Ha antibody when Ha-USP4 and Myc-IRF4 were coexpressed. (B) IRF4 was co-immunoprecipitated by the anti-Ha antibody when Ha-USP4 and Myc-IRF4 were coexpressed, and USP4 was co-immunoprecipitated by the anti-Myc antibody when Ha-USP4 and Myc-IRF4 were coexpressed. (C) IRF4 was co-immunoprecipitated by the anti-USP4 antibody, indicating that USP4 interacts with IRF4 in Th2 cells. Flag-IRF4 and Ha-USP4 were co-transfected into 293T cells. After 48 h, the cells were retrieved, and 1 µ g of anti-Ha or anti-Flag monoclonal antibody precipitate was added and immunoblotting was conducted in A and B. Th2 cells were amplified to 1×10 7 , and co-immunoprecipitated with 1 µ g of normal IgG or anti-USP4, then immunoblotting assay was conducted in C.

    Article Snippet: Cell culture 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT, USA) containing 10% FBS.

    Techniques: Immunoprecipitation, Transfection, Amplification

    M1 interacts with and does not disrupt procaspase-9–Apaf-1 interactions. Subconfluent 293T cellular monolayers were cotransfected with 500 ng C9DN-FLAG, 500 ng N-Apaf-1-myc, and 1,000 ng of either pCI, pM1L-V5, or pHA-K1L. At 24 h posttransfection, cells were lysed in NP-40 lysis buffer. Some lysates were set aside to monitor protein expression. For the remaining clarified cellular lysates, immunoprecipitations (IP) were performed using murine IgG or anti-FLAG (A), rabbit IgG or anti-V5 (B), or murine IgG or anti-HA antibodies (C) conjugated to protein G-Sepharose beads. Immunoprecipitated samples or 20 μg of cellular lysates was analyzed by SDS–8% PAGE, and proteins were transferred to a PVDF membrane for immunoblotting. Membranes were probed with the indicated antibodies to detect the epitope-tagged versions of C9DN, N-Apaf-1, M1, K1 or cellular actin. For panel B, all immunoprecipitated and cellular lysates were analyzed in the same gel and gels were spliced for labeling purposes.

    Journal: Journal of Virology

    Article Title: Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome

    doi: 10.1128/JVI.01385-17

    Figure Lengend Snippet: M1 interacts with and does not disrupt procaspase-9–Apaf-1 interactions. Subconfluent 293T cellular monolayers were cotransfected with 500 ng C9DN-FLAG, 500 ng N-Apaf-1-myc, and 1,000 ng of either pCI, pM1L-V5, or pHA-K1L. At 24 h posttransfection, cells were lysed in NP-40 lysis buffer. Some lysates were set aside to monitor protein expression. For the remaining clarified cellular lysates, immunoprecipitations (IP) were performed using murine IgG or anti-FLAG (A), rabbit IgG or anti-V5 (B), or murine IgG or anti-HA antibodies (C) conjugated to protein G-Sepharose beads. Immunoprecipitated samples or 20 μg of cellular lysates was analyzed by SDS–8% PAGE, and proteins were transferred to a PVDF membrane for immunoblotting. Membranes were probed with the indicated antibodies to detect the epitope-tagged versions of C9DN, N-Apaf-1, M1, K1 or cellular actin. For panel B, all immunoprecipitated and cellular lysates were analyzed in the same gel and gels were spliced for labeling purposes.

    Article Snippet: Human cervical carcinoma (HeLa) cells, human embryonic kidney 293T (293T) cells, and human acute monocytic leukemia (THP-1) cells were obtained from the American Type Culture Collection (ATCC).

    Techniques: Lysis, Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Labeling

    ZEB1 is a direct target of miR-342 in NPC. (A) Predicted human ZEB1 3′UTR binding site for miR-342. (B) A luciferase reporter assay was performed in 293T cells co-transfected with psiCHECK-ZEB1-3′UTR WT or psiCHECK-ZEB1-3′UTR MUT and miR-342 mimics or NC. *P

    Journal: Oncology Letters

    Article Title: MicroRNA-342 inhibits cell proliferation and invasion in nasopharyngeal carcinoma by directly targeting ZEB1

    doi: 10.3892/ol.2018.8788

    Figure Lengend Snippet: ZEB1 is a direct target of miR-342 in NPC. (A) Predicted human ZEB1 3′UTR binding site for miR-342. (B) A luciferase reporter assay was performed in 293T cells co-transfected with psiCHECK-ZEB1-3′UTR WT or psiCHECK-ZEB1-3′UTR MUT and miR-342 mimics or NC. *P

    Article Snippet: The human NPC SUNE1, 5–8F and 293T cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

    Techniques: Binding Assay, Luciferase, Reporter Assay, Transfection

    Retroviral expressions of calpastatin, calpastatin domains, and calpain and effect on IL-6 production from fibroblasts. (A) Retroviral construction and scheme of the domain structure of calpastatin. Domains I and IV are abbreviated as dI and dIV, respectively. (B) Calpastatin- or calpain-GRV was transfected into phoenix 293T cells by the calcium phosphate method. Three days later, the lysates of these cells were subjected to western blot analysis. The blots were probed with anti-calpastatin and anti-calpain antibodies. (C–D) Control cells, or calpastatin- or calpain-overexpressing NIH-3T3 cells (5x10 5 cells/ml) were cultured with 1 µg/ml LPS (C) or 10 ng/ml IL-17 (D) for 24 hours. The culture supernatants were collected and IL-6 concentrations were measured by ELISA. Left panel of (C) shows mock vs. calpastatins (Dunnett's test, n = 5), and right panel shows mock vs. calpain (Steel's test, n = 5). Panel D shows the results of three independent experiments (Steel's test). *P

    Journal: PLoS ONE

    Article Title: Overexpression of a Minimal Domain of Calpastatin Suppresses IL-6 Production and Th17 Development via Reduced NF-?B and Increased STAT5 Signals

    doi: 10.1371/journal.pone.0027020

    Figure Lengend Snippet: Retroviral expressions of calpastatin, calpastatin domains, and calpain and effect on IL-6 production from fibroblasts. (A) Retroviral construction and scheme of the domain structure of calpastatin. Domains I and IV are abbreviated as dI and dIV, respectively. (B) Calpastatin- or calpain-GRV was transfected into phoenix 293T cells by the calcium phosphate method. Three days later, the lysates of these cells were subjected to western blot analysis. The blots were probed with anti-calpastatin and anti-calpain antibodies. (C–D) Control cells, or calpastatin- or calpain-overexpressing NIH-3T3 cells (5x10 5 cells/ml) were cultured with 1 µg/ml LPS (C) or 10 ng/ml IL-17 (D) for 24 hours. The culture supernatants were collected and IL-6 concentrations were measured by ELISA. Left panel of (C) shows mock vs. calpastatins (Dunnett's test, n = 5), and right panel shows mock vs. calpain (Steel's test, n = 5). Panel D shows the results of three independent experiments (Steel's test). *P

    Article Snippet: Phoenix 293T cells and NIH-3T3 cells ( ATCC ) were maintained in DMEM with 10% FCS, 10 U/ml penicillin, 10 µg/ml streptomycin, 50 µM 2-ME, and 20 mM HEPES.

    Techniques: Transfection, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

    Substitution of CDK9 serine 90 for alanine reduces CDK9 association with large P-TEFb complex. 293T cells were transfected with Flag-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1-expressing vectors. At 48 hrs posttransfection, the cells were lysed with low salt (large complex – LC) buffer followed by high salt buffer (small complex – SC). CDK9 expression was analyzed by Western blotting. ( A) Representative immunoblots. (B) Quantification of CDK9 in large (LC) and small (SC) complexes conducted in two independent experiments. (C) Co-immunoprecipitation analysis. 293T cells were transfected with Flag-tagged WT CDK9 and CDK9 S90A and also co-transfected with Cyclin T1 expressing vectors. At 48 hrs post-transfection, the cells were lysed; CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies, resolved on 10% SDS PAGE and analyzed by immunoblotting with antibodies for Hexim1, CDK9 and cyclin T1. Lane 1, mock-transfected control.

    Journal: Retrovirology

    Article Title: CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    doi: 10.1186/1742-4690-9-94

    Figure Lengend Snippet: Substitution of CDK9 serine 90 for alanine reduces CDK9 association with large P-TEFb complex. 293T cells were transfected with Flag-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1-expressing vectors. At 48 hrs posttransfection, the cells were lysed with low salt (large complex – LC) buffer followed by high salt buffer (small complex – SC). CDK9 expression was analyzed by Western blotting. ( A) Representative immunoblots. (B) Quantification of CDK9 in large (LC) and small (SC) complexes conducted in two independent experiments. (C) Co-immunoprecipitation analysis. 293T cells were transfected with Flag-tagged WT CDK9 and CDK9 S90A and also co-transfected with Cyclin T1 expressing vectors. At 48 hrs post-transfection, the cells were lysed; CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies, resolved on 10% SDS PAGE and analyzed by immunoblotting with antibodies for Hexim1, CDK9 and cyclin T1. Lane 1, mock-transfected control.

    Article Snippet: Materials 293T cells were purchased from ATCC (Manassas, VA).

    Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, SDS Page

    CDK2 KD inhibits HIV-1 transcription. (A ) Expression of CDK2 mRNA is reduced in CDK2 KD cells. Total RNA was extracted from 293T cells and 293T-CDK KD cells lysates, reverse transcribed and analyzed by real-time PCR. β-Actin was used as an internal control. Quantification is shown in triplicates. (B) Cell cycle analysis of 293T-59 cells. Cells were fixed with 70% ethanol, stained with Propidium Iodine and analyze by FACS. Quantification is shown in triplicates. (C) CDK2 protein expression is reduced in 293T-CDK KD cells. Lysates from 293T and 293T-CDK2 KD cells (lanes 1 and 2) were analyzed for CDK2 expression by western blotting. Quantification is shown for three independent experiments. (D) Inhibition of VSVG HIV-1 Luc replication. 293T and 293T-CDK2-KD cells were infected with VSVG-pNL4-3 Luc virus and luciferase activity was measured. MTT assay was used for the normalization. Quantification is shown for three independent experiments. (E) CDK2 protein expression is reduced in HeLa-CDK2 KD cells. CDK2, tubulin and CDK9 expression was analyzed in HeLa and HeLa-CDK2 KD cells (lanes 1 and 2) by western blotting. (D) Inhibition of VSVG HIV-1 Luc replication. Lane1, CDK2 expression was analyzed in HeLa and HeLa-CDK2 KD cells by Real-time PCR. 18S RNA was used as an internal control. Quantification is shown in triplicates. Lane 2, Luciferase expression of VSVG-HIV-1 Luc was analyzed in infected HeLa and HeLa -CDK2-KD cells at 48 hours post infection. MTT assay was performed for the normalization. Quantification is shown for three independent experiments.

    Journal: Retrovirology

    Article Title: CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    doi: 10.1186/1742-4690-9-94

    Figure Lengend Snippet: CDK2 KD inhibits HIV-1 transcription. (A ) Expression of CDK2 mRNA is reduced in CDK2 KD cells. Total RNA was extracted from 293T cells and 293T-CDK KD cells lysates, reverse transcribed and analyzed by real-time PCR. β-Actin was used as an internal control. Quantification is shown in triplicates. (B) Cell cycle analysis of 293T-59 cells. Cells were fixed with 70% ethanol, stained with Propidium Iodine and analyze by FACS. Quantification is shown in triplicates. (C) CDK2 protein expression is reduced in 293T-CDK KD cells. Lysates from 293T and 293T-CDK2 KD cells (lanes 1 and 2) were analyzed for CDK2 expression by western blotting. Quantification is shown for three independent experiments. (D) Inhibition of VSVG HIV-1 Luc replication. 293T and 293T-CDK2-KD cells were infected with VSVG-pNL4-3 Luc virus and luciferase activity was measured. MTT assay was used for the normalization. Quantification is shown for three independent experiments. (E) CDK2 protein expression is reduced in HeLa-CDK2 KD cells. CDK2, tubulin and CDK9 expression was analyzed in HeLa and HeLa-CDK2 KD cells (lanes 1 and 2) by western blotting. (D) Inhibition of VSVG HIV-1 Luc replication. Lane1, CDK2 expression was analyzed in HeLa and HeLa-CDK2 KD cells by Real-time PCR. 18S RNA was used as an internal control. Quantification is shown in triplicates. Lane 2, Luciferase expression of VSVG-HIV-1 Luc was analyzed in infected HeLa and HeLa -CDK2-KD cells at 48 hours post infection. MTT assay was performed for the normalization. Quantification is shown for three independent experiments.

    Article Snippet: Materials 293T cells were purchased from ATCC (Manassas, VA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Cycle Assay, Staining, FACS, Western Blot, Inhibition, Infection, Luciferase, Activity Assay, MTT Assay

    Stable expression of CDK2-directed shRNA induces 7SK RNA expression. (A-C) Effect of CDK2 KD on small and large P-TEFb complexes. Lysates from 293T and 293T-CDK2 KD cells sequentially extracted with low and high salt buffers, were analyzed by immunoblotting for CDK9, cyclin T1, Hexim 1, eIF-2α and RNAPII. Panels B and C show average results from three experiments. (D ) CDK9 inhibitor ARC prevents CDK9 with association with large P-TEFb complex. Lysates from 293T cells untreated or treated with 10 μM ARC and extracted with low salt buffer (10 mM) or high salt buffer (450 mM) were resolved on 10% SDS-PAGE and analyzed by immunoblotting for CDK9 and tubulin. Average CDK9 expression adjusted to tubulin from two separate experiments is shown. (E) CDK2 KD increases the amount of 7SK RNA associated with CDK9. RNA isolated from co-immunoprecipitates with anti-CDK9 antibodies or control IgGs from 239T or 293T-CDK2 KD whole cell lysates was reverse transcribed and analyzed by semi-quantitative (30 cycles, upper panel) or real-time PCR (lower panel). Results are presented as numbers of copies of 7SK RNA. (F) CDK2 KD increases total 7SK RNA amount. 7SK RNA was analyzed in whole cell lysates of 293T or 293T-CDK2 KD cells by real-time PCR using 7SK expression vector as control. Results are presented as numbers of copies of 7SK RNA. (G) CDK2 KD increases 7SK RNA in the large complex fraction. 7SK RNA was analyzed by real-time PCR using U6 RNA as reference. Quantification is shown in triplicates.

    Journal: Retrovirology

    Article Title: CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    doi: 10.1186/1742-4690-9-94

    Figure Lengend Snippet: Stable expression of CDK2-directed shRNA induces 7SK RNA expression. (A-C) Effect of CDK2 KD on small and large P-TEFb complexes. Lysates from 293T and 293T-CDK2 KD cells sequentially extracted with low and high salt buffers, were analyzed by immunoblotting for CDK9, cyclin T1, Hexim 1, eIF-2α and RNAPII. Panels B and C show average results from three experiments. (D ) CDK9 inhibitor ARC prevents CDK9 with association with large P-TEFb complex. Lysates from 293T cells untreated or treated with 10 μM ARC and extracted with low salt buffer (10 mM) or high salt buffer (450 mM) were resolved on 10% SDS-PAGE and analyzed by immunoblotting for CDK9 and tubulin. Average CDK9 expression adjusted to tubulin from two separate experiments is shown. (E) CDK2 KD increases the amount of 7SK RNA associated with CDK9. RNA isolated from co-immunoprecipitates with anti-CDK9 antibodies or control IgGs from 239T or 293T-CDK2 KD whole cell lysates was reverse transcribed and analyzed by semi-quantitative (30 cycles, upper panel) or real-time PCR (lower panel). Results are presented as numbers of copies of 7SK RNA. (F) CDK2 KD increases total 7SK RNA amount. 7SK RNA was analyzed in whole cell lysates of 293T or 293T-CDK2 KD cells by real-time PCR using 7SK expression vector as control. Results are presented as numbers of copies of 7SK RNA. (G) CDK2 KD increases 7SK RNA in the large complex fraction. 7SK RNA was analyzed by real-time PCR using U6 RNA as reference. Quantification is shown in triplicates.

    Article Snippet: Materials 293T cells were purchased from ATCC (Manassas, VA).

    Techniques: Expressing, shRNA, RNA Expression, SDS Page, Isolation, Real-time Polymerase Chain Reaction, Plasmid Preparation

    CDK2 phosphorylates Ser90 residue of CDK9. (A) CDK2 phosphorylates Ser90-containing peptide. CDK9-derived chemically synthesized peptides containing Thr29; Ser175 and Thr186; or Ser90 residues were phosphorylated by recombinant CDK2/cyclin E and analyzed by Phosphor Imaging Device (upper panel) or stained with Coomassie (lower panel). (B) CDK9 S90A mutant is less phosphorylated by CDK2/cyclin E in vitro . WT CDK9 and CDK9 S90A were expressed in 293T cells, precipitated with anti-FLAG antibodies and incubated with recombinant CDK2/cyclin E in the presence of ( 32 P) ATP. The reactions were resolved on a 10% SDS Tris-glycine gel and analyzed by immunoblotting (upper panel) or on Phosphor Imaging Device (lower panel). Quantification from the Phosphor Imager is shown. (C) Substitution of CDK9 Ser 90 prevents its phosphorylation in cultured cells. 293T cells transfected with FLAG-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1, pulse-labeled with ( 32 P) and CDK9 was immunoprecipitated from cellular lysates with anti-FLAG antibodies, and analyzed by immunoblotting (upper panel) or by Phosphor Imaging Device (lower panel). Quantification of the bands on Phosphor Imager is shown for three independent experiments. (D and E) CDK9 Ser90 phosphorylation is decreased in CDK2 KD cells. 293T cells were transfected with vectors expressing Flag-CDK9 WT or Flag-CDK9 S175A (panel D); or 293T and 293T-59 cells were transfected with a vector expressing Flag-CDK9 WT (panel E) and after 48 hrs in culture treated with 0.1 μM okadaic acid. CDK9 was immunoprecipitated with anti-Flag antibodies and analyzed by immunoblotting with anti-CDK9 or Ser90 phospho-specific antibodies.

    Journal: Retrovirology

    Article Title: CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    doi: 10.1186/1742-4690-9-94

    Figure Lengend Snippet: CDK2 phosphorylates Ser90 residue of CDK9. (A) CDK2 phosphorylates Ser90-containing peptide. CDK9-derived chemically synthesized peptides containing Thr29; Ser175 and Thr186; or Ser90 residues were phosphorylated by recombinant CDK2/cyclin E and analyzed by Phosphor Imaging Device (upper panel) or stained with Coomassie (lower panel). (B) CDK9 S90A mutant is less phosphorylated by CDK2/cyclin E in vitro . WT CDK9 and CDK9 S90A were expressed in 293T cells, precipitated with anti-FLAG antibodies and incubated with recombinant CDK2/cyclin E in the presence of ( 32 P) ATP. The reactions were resolved on a 10% SDS Tris-glycine gel and analyzed by immunoblotting (upper panel) or on Phosphor Imaging Device (lower panel). Quantification from the Phosphor Imager is shown. (C) Substitution of CDK9 Ser 90 prevents its phosphorylation in cultured cells. 293T cells transfected with FLAG-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1, pulse-labeled with ( 32 P) and CDK9 was immunoprecipitated from cellular lysates with anti-FLAG antibodies, and analyzed by immunoblotting (upper panel) or by Phosphor Imaging Device (lower panel). Quantification of the bands on Phosphor Imager is shown for three independent experiments. (D and E) CDK9 Ser90 phosphorylation is decreased in CDK2 KD cells. 293T cells were transfected with vectors expressing Flag-CDK9 WT or Flag-CDK9 S175A (panel D); or 293T and 293T-59 cells were transfected with a vector expressing Flag-CDK9 WT (panel E) and after 48 hrs in culture treated with 0.1 μM okadaic acid. CDK9 was immunoprecipitated with anti-Flag antibodies and analyzed by immunoblotting with anti-CDK9 or Ser90 phospho-specific antibodies.

    Article Snippet: Materials 293T cells were purchased from ATCC (Manassas, VA).

    Techniques: Derivative Assay, Synthesized, Recombinant, Imaging, Staining, Mutagenesis, In Vitro, Incubation, Cell Culture, Transfection, Labeling, Immunoprecipitation, Expressing, Plasmid Preparation

    CDK9 Ser 90 substitutions affect Tat-dependent HIV-1 transcription. (A-B) 293T cells were transfected with HIV-1 LTR-Luc expression vector along with WT CDK9, CDK9 S90A and CDK9 S90D expression vectors without (panel A) or with Tat expression vector (panel B). Cells were lysed at 24 hours posttransfection and luciferase activity was measured followed by the measurement of EGFP fluorescence, which was used for the normalization. Quantification is shown for three independent experiments. (C) Expression of CDK9 and cyclin T1. To determine the levels of CDK9 and cyclin T1 expression, 293T cells were transfected with FLAG-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1 and HIV-1 Tat expression vectors. At 48 hrs post-transfection, the cells were lysed, the lysates were resolved on 10% SDS PAGE and analyzed by immunoblotting with antibodies for cyclin T1, CDK9, and tubulin was used as loading control. Lane 1, mock-transfected control.

    Journal: Retrovirology

    Article Title: CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    doi: 10.1186/1742-4690-9-94

    Figure Lengend Snippet: CDK9 Ser 90 substitutions affect Tat-dependent HIV-1 transcription. (A-B) 293T cells were transfected with HIV-1 LTR-Luc expression vector along with WT CDK9, CDK9 S90A and CDK9 S90D expression vectors without (panel A) or with Tat expression vector (panel B). Cells were lysed at 24 hours posttransfection and luciferase activity was measured followed by the measurement of EGFP fluorescence, which was used for the normalization. Quantification is shown for three independent experiments. (C) Expression of CDK9 and cyclin T1. To determine the levels of CDK9 and cyclin T1 expression, 293T cells were transfected with FLAG-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1 and HIV-1 Tat expression vectors. At 48 hrs post-transfection, the cells were lysed, the lysates were resolved on 10% SDS PAGE and analyzed by immunoblotting with antibodies for cyclin T1, CDK9, and tubulin was used as loading control. Lane 1, mock-transfected control.

    Article Snippet: Materials 293T cells were purchased from ATCC (Manassas, VA).

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

    Inhibition of CDK9 activity and phosphorylation by CDK2-directed siRNA. (A-B) Inhibition of CDK2 expression and CDK9 activity. Lysates from 293T cells transfected with CDK2-directed (lane 1) or control siRNA (lane 2) were analyzed by immunoblotting for the expression of CDK2, CDK9, cyclin T1 and tubulin (panel A)Panel B, the cells were also co-transfected with Flag-CDK9 and cyclin T1 expression vectors. Lysates were immunoblotted with antibodies against CDK2, Flag and α-tubulin as loading control (upper panel) or immunoprecipitated with anti-Flag antibodies (lanes 1 and 2) or non-specific IgGs (lane 3) (lower panel). CDK9 activity was analyzed with GST-CTD as substrate. Lane 4, control recombinant CDK9/cyclin T1. GST-CTD is shown as Coomassie stain. Quantification is shown as average from three independent experiments. (C) Inhibition of CDK9 phosphorylation. Lysates from 293T cells transfected with CDK2-directed (lane 3) or control siRNA (lanes 1 and 2) and also with a vector expressing Flag-CDK9 WT as in panel B then pulse-labeled with ( 32 P) were immunoprecipitated with anti-Flag antibodies (lanes 2 and 3) or with non-specific IgGs (lane 1), resolved on 10% SDS PAGE and exposed to a Phosphor imaging device (upper panel) or analyzed by immunoblotting with anti-CDK9 antibodies (lower panel). (D) No effect on CDK9 Thr186 phosphorylation. Lysates from 293T cells transfected CDK2-directed (lane 1) or control siRNA (lanes 2 and 3) and then co-transfected with vectors expressing Flag-CDK9 WT (lanes 1 and 2) or Flag-CDK9 T186A (lane 3) were immunoprecipitated with anti-Flag antibodies and analyzed by immunoblotting with Thr186 phospho-specific or anti-CDK9 antibodies.

    Journal: Retrovirology

    Article Title: CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    doi: 10.1186/1742-4690-9-94

    Figure Lengend Snippet: Inhibition of CDK9 activity and phosphorylation by CDK2-directed siRNA. (A-B) Inhibition of CDK2 expression and CDK9 activity. Lysates from 293T cells transfected with CDK2-directed (lane 1) or control siRNA (lane 2) were analyzed by immunoblotting for the expression of CDK2, CDK9, cyclin T1 and tubulin (panel A)Panel B, the cells were also co-transfected with Flag-CDK9 and cyclin T1 expression vectors. Lysates were immunoblotted with antibodies against CDK2, Flag and α-tubulin as loading control (upper panel) or immunoprecipitated with anti-Flag antibodies (lanes 1 and 2) or non-specific IgGs (lane 3) (lower panel). CDK9 activity was analyzed with GST-CTD as substrate. Lane 4, control recombinant CDK9/cyclin T1. GST-CTD is shown as Coomassie stain. Quantification is shown as average from three independent experiments. (C) Inhibition of CDK9 phosphorylation. Lysates from 293T cells transfected with CDK2-directed (lane 3) or control siRNA (lanes 1 and 2) and also with a vector expressing Flag-CDK9 WT as in panel B then pulse-labeled with ( 32 P) were immunoprecipitated with anti-Flag antibodies (lanes 2 and 3) or with non-specific IgGs (lane 1), resolved on 10% SDS PAGE and exposed to a Phosphor imaging device (upper panel) or analyzed by immunoblotting with anti-CDK9 antibodies (lower panel). (D) No effect on CDK9 Thr186 phosphorylation. Lysates from 293T cells transfected CDK2-directed (lane 1) or control siRNA (lanes 2 and 3) and then co-transfected with vectors expressing Flag-CDK9 WT (lanes 1 and 2) or Flag-CDK9 T186A (lane 3) were immunoprecipitated with anti-Flag antibodies and analyzed by immunoblotting with Thr186 phospho-specific or anti-CDK9 antibodies.

    Article Snippet: Materials 293T cells were purchased from ATCC (Manassas, VA).

    Techniques: Inhibition, Activity Assay, Expressing, Transfection, Immunoprecipitation, Recombinant, Staining, Plasmid Preparation, Labeling, SDS Page, Imaging

    S79A mutation impairs the interaction of PAK1 with Cdc42 and Rac1. ( A ) Interaction between Myc-Cdc42 and GFP-PAK1. GFP-PAK1 WT or GFP-PAK1 S79A was coexpressed with Myc-Cdc42 in 293T cells. Cell extracts were immunoprecipitated with anti-GFP antibody (IP) and then immunoblotted with anti-GFP or anti-Myc antibody (left). ( B ) Interaction between GFP-Rac1 and Myc-PAK1. Cell extracts were immunoprecipitated with anti-Myc antibody (IP) and then immunoblotted with anti-Myc or anti-GFP antibody. ( C and D ) Cell lysates prepared from 293T cells expressing GFP- PAK1 WT or GFP- PAK1 S79A were incubated with GST-Cdc42 (C) or GST-Rac1 (D) bound to GST-beads. Upper panels, Western blot analysis of the eluates from the beads using anti-GFP antibody; Lower panels, the blots were stained with Ponceau S Stain. MW markers; molecular weight markers.

    Journal: PLoS ONE

    Article Title: Evidence for a Novel Mechanism of the PAK1 Interaction with the Rho-GTPases Cdc42 and Rac

    doi: 10.1371/journal.pone.0071495

    Figure Lengend Snippet: S79A mutation impairs the interaction of PAK1 with Cdc42 and Rac1. ( A ) Interaction between Myc-Cdc42 and GFP-PAK1. GFP-PAK1 WT or GFP-PAK1 S79A was coexpressed with Myc-Cdc42 in 293T cells. Cell extracts were immunoprecipitated with anti-GFP antibody (IP) and then immunoblotted with anti-GFP or anti-Myc antibody (left). ( B ) Interaction between GFP-Rac1 and Myc-PAK1. Cell extracts were immunoprecipitated with anti-Myc antibody (IP) and then immunoblotted with anti-Myc or anti-GFP antibody. ( C and D ) Cell lysates prepared from 293T cells expressing GFP- PAK1 WT or GFP- PAK1 S79A were incubated with GST-Cdc42 (C) or GST-Rac1 (D) bound to GST-beads. Upper panels, Western blot analysis of the eluates from the beads using anti-GFP antibody; Lower panels, the blots were stained with Ponceau S Stain. MW markers; molecular weight markers.

    Article Snippet: RWPE-1, PC3, and 293T cells cell lines used in this study were obtained from the American Type Culture Collection (ATCC; Rockville, MD).

    Techniques: Mutagenesis, Immunoprecipitation, Expressing, Incubation, Western Blot, Staining, Molecular Weight

    R179X impact on protein allele expression and cellular localization. ( a ) Schematic diagram of the RNF186 protein with a zinc finger RING-type and two helical transmembrane (TM1 and TM2) domains, and the A64T and R179X variants shown. ( b ) 293T cells were transfected with the indicated expression constructs and analysed by western blot for expression of Rnf186 and the R179X variant. The RNF186 protein with a premature stop at amino-acid position 179 is expressed, but at reduced levels. ( c ) 293T cells were transfected with the indicated expression constructs and analysed by immunofluorescence to demonstrate altered subcellular localization of the R179X variant.

    Journal: Nature Communications

    Article Title: A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis

    doi: 10.1038/ncomms12342

    Figure Lengend Snippet: R179X impact on protein allele expression and cellular localization. ( a ) Schematic diagram of the RNF186 protein with a zinc finger RING-type and two helical transmembrane (TM1 and TM2) domains, and the A64T and R179X variants shown. ( b ) 293T cells were transfected with the indicated expression constructs and analysed by western blot for expression of Rnf186 and the R179X variant. The RNF186 protein with a premature stop at amino-acid position 179 is expressed, but at reduced levels. ( c ) 293T cells were transfected with the indicated expression constructs and analysed by immunofluorescence to demonstrate altered subcellular localization of the R179X variant.

    Article Snippet: Biochemistry 293T cells (American Type Culture Collection) were transfected with RNF186 expression constructs by means of Lipofectamine 2000 (Life Technologies) as indicated by the manufacturer.

    Techniques: Expressing, Transfection, Construct, Western Blot, Variant Assay, Immunofluorescence

    HPLC purification of in vitro transcribed mRNA enhances translation. 293T ( A ) and human DCs ( B and C ) were transfected with TransIT- (A and C) or Lipofectin- (B) complexed TEVRenA 51 or TEVmEPOA 51 mRNA with the indicated modifications with or without HPLC purification and analyzed for Renilla luciferase activity or levels of supernatant-associated mEPO protein at 24 h. ( D ) Human DCs were transfected with Ψ-modified TEVeGFPA n mRNA with or without HPLC purification (0.1 µg/well) complexed with Lipofectin or TransIT and analyzed 24 h later. Error bars are standard error of the mean. Data shown is from one experiment that is representative of three or more.

    Journal: Nucleic Acids Research

    Article Title: Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA

    doi: 10.1093/nar/gkr695

    Figure Lengend Snippet: HPLC purification of in vitro transcribed mRNA enhances translation. 293T ( A ) and human DCs ( B and C ) were transfected with TransIT- (A and C) or Lipofectin- (B) complexed TEVRenA 51 or TEVmEPOA 51 mRNA with the indicated modifications with or without HPLC purification and analyzed for Renilla luciferase activity or levels of supernatant-associated mEPO protein at 24 h. ( D ) Human DCs were transfected with Ψ-modified TEVeGFPA n mRNA with or without HPLC purification (0.1 µg/well) complexed with Lipofectin or TransIT and analyzed 24 h later. Error bars are standard error of the mean. Data shown is from one experiment that is representative of three or more.

    Article Snippet: Cells Human embryonic kidney 293T cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM l -glutamine (Life Technologies) and 10% fetal calf serum (FCS) (HyClone) (complete medium).

    Techniques: High Performance Liquid Chromatography, Purification, In Vitro, Transfection, Luciferase, Activity Assay, Modification

    Construction and in vitro activity of ΔLMP1-MAVS. (A) Cloning strategy for vectors expressing full length LMP1 or ΔLMP1-MAVS fusion. (B) Western blot analysis of lysate from 293T cells transfected with ΔLMP1-MAVS plasmid. Luciferase activity assay was performed on 293T cells co-transfected with NF-κB (C) or IFN-β (D) luciferase reporter constructs and constructs expressing LMP1, ΔLMP1-MAVS, Flag-TRAF6, or ΔRIG-I. Fold induction was measured relative to empty vector pcDNA3.1. *p

    Journal: PLoS ONE

    Article Title: Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    doi: 10.1371/journal.pone.0148929

    Figure Lengend Snippet: Construction and in vitro activity of ΔLMP1-MAVS. (A) Cloning strategy for vectors expressing full length LMP1 or ΔLMP1-MAVS fusion. (B) Western blot analysis of lysate from 293T cells transfected with ΔLMP1-MAVS plasmid. Luciferase activity assay was performed on 293T cells co-transfected with NF-κB (C) or IFN-β (D) luciferase reporter constructs and constructs expressing LMP1, ΔLMP1-MAVS, Flag-TRAF6, or ΔRIG-I. Fold induction was measured relative to empty vector pcDNA3.1. *p

    Article Snippet: Cells, Viruses and Reagents 293T cells (American Type Culture Collection, Manassas, VA) were cultured in complete DMEM medium (Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone Inc.), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin).

    Techniques: In Vitro, Activity Assay, Clone Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Luciferase, Construct

    Gene array analysis of transfected 293T cells and primary CD4+ T cells cultured with 293T supernatant. Three independent wells of 293T cells were transfected with pcDNA3.1 empty vector or ΔLMP1-MAVS plasmid and total RNA isolated 36 hours later. Primary CD4+ T cells from 3 independent donors were cultured with 293T supernatant (collected 24 hours following pcDNA3.1 or ΔLMP1-MAVS transfection). Total CD4+ T cell RNA was isolated 36 hours later. (A) Venn Diagrams of the number of probe sets upregulated and down-regulated ( > 2-fold change) by ΔLMP1-MAVS. (B) Differential gene expression of 293T cells and CD4+ T cells. (C) List of genes upregulated by ΔLMP1-MAVS in both 293T cells and CD4+ T cells. Fold-change and P-values for each probe set are shown for CD4+ T cells following ΔLMP1-MAVS treatment. (D) List of genes upregulated by CD4+ T cells but not 293T cells. Fold-change between pcDNA3.1 and pΔLMP1-MAVS and P-values for each probe set are shown. (E) Gen Go networks analysis of pathways significantly upregulated by ΔLMP1-MAVS in transfected 293T cells, or CD4+ T cells cultured with ΔLMP1-MAVS transfected 293T supernatant.

    Journal: PLoS ONE

    Article Title: Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    doi: 10.1371/journal.pone.0148929

    Figure Lengend Snippet: Gene array analysis of transfected 293T cells and primary CD4+ T cells cultured with 293T supernatant. Three independent wells of 293T cells were transfected with pcDNA3.1 empty vector or ΔLMP1-MAVS plasmid and total RNA isolated 36 hours later. Primary CD4+ T cells from 3 independent donors were cultured with 293T supernatant (collected 24 hours following pcDNA3.1 or ΔLMP1-MAVS transfection). Total CD4+ T cell RNA was isolated 36 hours later. (A) Venn Diagrams of the number of probe sets upregulated and down-regulated ( > 2-fold change) by ΔLMP1-MAVS. (B) Differential gene expression of 293T cells and CD4+ T cells. (C) List of genes upregulated by ΔLMP1-MAVS in both 293T cells and CD4+ T cells. Fold-change and P-values for each probe set are shown for CD4+ T cells following ΔLMP1-MAVS treatment. (D) List of genes upregulated by CD4+ T cells but not 293T cells. Fold-change between pcDNA3.1 and pΔLMP1-MAVS and P-values for each probe set are shown. (E) Gen Go networks analysis of pathways significantly upregulated by ΔLMP1-MAVS in transfected 293T cells, or CD4+ T cells cultured with ΔLMP1-MAVS transfected 293T supernatant.

    Article Snippet: Cells, Viruses and Reagents 293T cells (American Type Culture Collection, Manassas, VA) were cultured in complete DMEM medium (Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone Inc.), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin).

    Techniques: Transfection, Cell Culture, Plasmid Preparation, Isolation, Expressing

    Inhibition of HIV and SIV viral infection of TZM-bl and primary human CD4+ T cells. The relative level of viral replication in TZM-bl cells was measured following incubation with supernatant from 293T cells transfected with pcDNA3.1 vector expressing GFP (control), LMP1, or ΔLMP1-MAVS plasmid. (A) Infection with HIV-1 BaL strain. (B) Infection with VSV-G pseudotyped single cycle SIV. (C) CD4+ T cells were isolated from a healthy donor by negative selection, activated, and cultured with 293T supernatant for 24 hours. Cells were then washed and infected with HIV-1 BaL at an MOI or 0.1 or 1. The concentration of p24 was measured 6 days later by ELISA assay. *p

    Journal: PLoS ONE

    Article Title: Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    doi: 10.1371/journal.pone.0148929

    Figure Lengend Snippet: Inhibition of HIV and SIV viral infection of TZM-bl and primary human CD4+ T cells. The relative level of viral replication in TZM-bl cells was measured following incubation with supernatant from 293T cells transfected with pcDNA3.1 vector expressing GFP (control), LMP1, or ΔLMP1-MAVS plasmid. (A) Infection with HIV-1 BaL strain. (B) Infection with VSV-G pseudotyped single cycle SIV. (C) CD4+ T cells were isolated from a healthy donor by negative selection, activated, and cultured with 293T supernatant for 24 hours. Cells were then washed and infected with HIV-1 BaL at an MOI or 0.1 or 1. The concentration of p24 was measured 6 days later by ELISA assay. *p

    Article Snippet: Cells, Viruses and Reagents 293T cells (American Type Culture Collection, Manassas, VA) were cultured in complete DMEM medium (Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone Inc.), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin).

    Techniques: Inhibition, Infection, Incubation, Transfection, Plasmid Preparation, Expressing, Isolation, Selection, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Exosome-depleted 293T supernatant inhibits HIV and SIV replication. 293T cells were transfected with pcDNA3.1 plasmids encoding GFP, LMP1, or ΔLMP1-MAVS and supernatant was isolated. Supernatant was depleted of exosomes by ultracentrifugation. (A) TZM-bl cells were cultured with isolated exosomes and infected with increasing concentrations of HIV-1 BaL. (B) TZM-bl cells were cultured with exosome-depleted supernatant, followed by infection with HIV-1 BaL. (C) TZM-bl cells were cultured with exosome-depleted supernatant, followed by infection with VSV-G pseudotyped single cycle SIV. *p

    Journal: PLoS ONE

    Article Title: Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    doi: 10.1371/journal.pone.0148929

    Figure Lengend Snippet: Exosome-depleted 293T supernatant inhibits HIV and SIV replication. 293T cells were transfected with pcDNA3.1 plasmids encoding GFP, LMP1, or ΔLMP1-MAVS and supernatant was isolated. Supernatant was depleted of exosomes by ultracentrifugation. (A) TZM-bl cells were cultured with isolated exosomes and infected with increasing concentrations of HIV-1 BaL. (B) TZM-bl cells were cultured with exosome-depleted supernatant, followed by infection with HIV-1 BaL. (C) TZM-bl cells were cultured with exosome-depleted supernatant, followed by infection with VSV-G pseudotyped single cycle SIV. *p

    Article Snippet: Cells, Viruses and Reagents 293T cells (American Type Culture Collection, Manassas, VA) were cultured in complete DMEM medium (Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone Inc.), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin).

    Techniques: Transfection, Isolation, Cell Culture, Infection

    RealTime PCR analysis of 293T cells transfected with LMP1 or ΔLMP1-MAVS. 293T cells were transfected with pcDNA3.1, LMP1 plasmid, or ΔLMP1-MAVS plasmid and total RNA isolated following 36-hour culture. Normalized expression was determined relative to the pcDNA3.1 control.

    Journal: PLoS ONE

    Article Title: Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    doi: 10.1371/journal.pone.0148929

    Figure Lengend Snippet: RealTime PCR analysis of 293T cells transfected with LMP1 or ΔLMP1-MAVS. 293T cells were transfected with pcDNA3.1, LMP1 plasmid, or ΔLMP1-MAVS plasmid and total RNA isolated following 36-hour culture. Normalized expression was determined relative to the pcDNA3.1 control.

    Article Snippet: Cells, Viruses and Reagents 293T cells (American Type Culture Collection, Manassas, VA) were cultured in complete DMEM medium (Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone Inc.), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin).

    Techniques: Polymerase Chain Reaction, Transfection, Plasmid Preparation, Isolation, Expressing

    Beta-interferon mediates inhibition of HIV-1 BaL replication. (A) The relative level of HIV-1 BaL strain viral replication in TZM-bl cells was measured following incubation with supernatant from 293T cells transfected with either pcDNA3.1 or pΔLMP1-MAVS plasmid, combined with 60 μg of isotype control antibody, anti-IFN-β antibody, or anti-IFN-α antibody. (B) Human CD4+ T cells were infected with HIV-1 BaL in the presence of increasing concentrations of interferon-α and compared to infection in the presence of 293T supernatant following transfection with either pcDNA3.1 or pΔLMP1-MAVS plasmid. (C) Supernatant from 293T cells transfected with pcDNA3.1 GFP, LMP1, or ΔLMP1-MAVS plasmid was assayed for IFN-α and IFN-β secretion by ELISA. NT: no treatment.

    Journal: PLoS ONE

    Article Title: Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    doi: 10.1371/journal.pone.0148929

    Figure Lengend Snippet: Beta-interferon mediates inhibition of HIV-1 BaL replication. (A) The relative level of HIV-1 BaL strain viral replication in TZM-bl cells was measured following incubation with supernatant from 293T cells transfected with either pcDNA3.1 or pΔLMP1-MAVS plasmid, combined with 60 μg of isotype control antibody, anti-IFN-β antibody, or anti-IFN-α antibody. (B) Human CD4+ T cells were infected with HIV-1 BaL in the presence of increasing concentrations of interferon-α and compared to infection in the presence of 293T supernatant following transfection with either pcDNA3.1 or pΔLMP1-MAVS plasmid. (C) Supernatant from 293T cells transfected with pcDNA3.1 GFP, LMP1, or ΔLMP1-MAVS plasmid was assayed for IFN-α and IFN-β secretion by ELISA. NT: no treatment.

    Article Snippet: Cells, Viruses and Reagents 293T cells (American Type Culture Collection, Manassas, VA) were cultured in complete DMEM medium (Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone Inc.), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin).

    Techniques: Inhibition, Incubation, Transfection, Plasmid Preparation, Infection, Enzyme-linked Immunosorbent Assay

    Stability of gp160 is unchanged in Ubc9 knockdown cells. (a) 293T cells were transfected with pNL4-3 MUT 511 alone, or in combination with Ctr. siRNA or Ubc9 siRNA, or left untransfected. Cells were pulse (P) labeled with [ 35 S] methionine/cysteine for 1 hour, then chased for 2 and 4 hours. Samples were processed as described in previous Endo H f experiment. A representative over-exposed gel is shown so that partially Endo H f resistant Env can be more easily visualized. The identity of Endo H f , untreated viral proteins and their positions in the gel are labeled on the right. Deglycosylated Endo H f sensitive forms of gp160 residing in the ER are labeled as gp160s. Partially deglycosylated, Endo H f resistant forms of gp160 that have undergone glycan modification in the TGN are labeled as gp160r. These forms of gp160 denoted as gp160* in Endo H f untreated samples. Lower panel: Immunoblot of Ubc9 expression in whole cell lysates; untransfected (group 1), pNL4-3 (group 2), Ctr. siRNA (group 3), and Ubc9 siRNA (group 4). (b) Furin activity is not dependent upon Ubc9 expression. 293T cells were lysed 24 hrs post transfection and cell lysates were immunoblotted with antibodies against Ubc9, Actin, and E-Cadherin. Immature, uncleaved E-Cadherin is designated as pro E-Cadherin.

    Journal: PLoS ONE

    Article Title: Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner

    doi: 10.1371/journal.pone.0069359

    Figure Lengend Snippet: Stability of gp160 is unchanged in Ubc9 knockdown cells. (a) 293T cells were transfected with pNL4-3 MUT 511 alone, or in combination with Ctr. siRNA or Ubc9 siRNA, or left untransfected. Cells were pulse (P) labeled with [ 35 S] methionine/cysteine for 1 hour, then chased for 2 and 4 hours. Samples were processed as described in previous Endo H f experiment. A representative over-exposed gel is shown so that partially Endo H f resistant Env can be more easily visualized. The identity of Endo H f , untreated viral proteins and their positions in the gel are labeled on the right. Deglycosylated Endo H f sensitive forms of gp160 residing in the ER are labeled as gp160s. Partially deglycosylated, Endo H f resistant forms of gp160 that have undergone glycan modification in the TGN are labeled as gp160r. These forms of gp160 denoted as gp160* in Endo H f untreated samples. Lower panel: Immunoblot of Ubc9 expression in whole cell lysates; untransfected (group 1), pNL4-3 (group 2), Ctr. siRNA (group 3), and Ubc9 siRNA (group 4). (b) Furin activity is not dependent upon Ubc9 expression. 293T cells were lysed 24 hrs post transfection and cell lysates were immunoblotted with antibodies against Ubc9, Actin, and E-Cadherin. Immature, uncleaved E-Cadherin is designated as pro E-Cadherin.

    Article Snippet: Cell culture and transfection 293T cells were obtained from the American Type Culture Collection, they were cultured and transfected with control and Ubc9 siRNA as previously reported [ ].

    Techniques: Transfection, Labeling, Modification, Expressing, Activity Assay

    Physical interaction between Dnmt1 and the MutSα complex. ( A ) The potential for physical interaction between Dnmt1 and MutSα complex was examined by immunoprecipitation (IP) coupled with Western blotting (IB). Extracts from 293T cells co-expressing Dnmt1, HA-Msh6 and Flag-Msh2 were immunoprecipitated with control rabbit IgG, mouse IgG, anti-Dnmt1, anti-HA or anti-Flag, and then analyzed by IB using the latter three antibodies. The detection by IB of HA-Msh6 and Flag-Msh2 in the anti-Dnmt1 precipitate, as well as detection of Dnmt1 in the anti-HA (Msh6) or anti-Flag (Msh2) precipitate, indicates that Dnmt1 physically interacts with the MutSα complex. ( B ) Domain(s) of Dnmt1 interacting with the MutSα complex. The domain(s) was mapped by IP/IB assay of extracts from 293T cells co-expressing HA-Msh6, Flag-Msh2, and different fragments of Dnmt1. As summarized in the diagram, the IP/IB data (see Supplementary Fig. S4 ) show that Dnmt1 interacts with the MutSα complex through its central region (a.a. 446~1080), which is non-overlapping with the Np95-interacting domains of Dnmt1.

    Journal: Scientific Reports

    Article Title: Epigenetic Enhancement of the Post-replicative DNA Mismatch Repair of Mammalian Genomes by a Hemi-mCpG-Np95-Dnmt1 Axis

    doi: 10.1038/srep37490

    Figure Lengend Snippet: Physical interaction between Dnmt1 and the MutSα complex. ( A ) The potential for physical interaction between Dnmt1 and MutSα complex was examined by immunoprecipitation (IP) coupled with Western blotting (IB). Extracts from 293T cells co-expressing Dnmt1, HA-Msh6 and Flag-Msh2 were immunoprecipitated with control rabbit IgG, mouse IgG, anti-Dnmt1, anti-HA or anti-Flag, and then analyzed by IB using the latter three antibodies. The detection by IB of HA-Msh6 and Flag-Msh2 in the anti-Dnmt1 precipitate, as well as detection of Dnmt1 in the anti-HA (Msh6) or anti-Flag (Msh2) precipitate, indicates that Dnmt1 physically interacts with the MutSα complex. ( B ) Domain(s) of Dnmt1 interacting with the MutSα complex. The domain(s) was mapped by IP/IB assay of extracts from 293T cells co-expressing HA-Msh6, Flag-Msh2, and different fragments of Dnmt1. As summarized in the diagram, the IP/IB data (see Supplementary Fig. S4 ) show that Dnmt1 interacts with the MutSα complex through its central region (a.a. 446~1080), which is non-overlapping with the Np95-interacting domains of Dnmt1.

    Article Snippet: The 293T cell line was obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium plus 10% fetal bovine serum.

    Techniques: Immunoprecipitation, Western Blot, Expressing

    The Retrovolution system. Panel A . General structure of genomic RNAs for Retrovolution (top) and specific structure of the genomic RNA used in this study (bottom). Expression of the “target gene” is driven by an eukaryotic promoter (inducible or not, in this case the constitutive promoter of the human elongation factor 1-alpha, EF1-alpha); a selectable marker (here, the puromycin-resistance gene) is transcribed from a second, independent promoter (in this case the human phosphoglycerate kinase gene, hPGK) situated immediately downstream of the translation termination signal of the first gene. U3, R, U5 (LTRs) and HIV-1 cis-acting sequences are viral sequences necessary for viral genome packaging, reverse-transcription and integration. Panel B . Experimental procedure of Retrovolution. White arrows indicate the initial steps of the procedure, lilac arrows the steps involved in the generation of genetic diversity, and green arrows those involved in the selection steps. (a) HIV-based VSV-pseudotyped defective vectors are generated by transfection of producer cells (HEK-293T cells) with two plasmids leading to the production of the viral proteins (transcomplementation plasmids, pHCMV-G and pCMVΔR8.91), and one or potentially more plasmids leading to the synthesis of the genomic RNA (pSDY-dCK-Puro, outlined in panel A). The retroviral vectors generated (parental generation) are used to transduce (b) producer cells (HEK-293T) at a high MOI. Expression of the puromycin-resistance gene from proviral DNA allows the selection of the efficiently transduced cell population (c). Upon transfection of these cells with transcomplementation plasmids (d), new particles encapsidating the gRNA transcribed from the proviral DNA inserted in the cell's genome will be generated and used to transduce fresh producer cells (e). Steps (c), (d), and (e) are reiterated, producing subsequent viral generations (each cycle requires a week in the case of puromycin selection, but this time varies with the selectable marker used). To screen the library, some of the vectors are used to transduce the appropriate target cells at a low MOI (f). Transduced cells can then be directly screened on the basis of the desired phenotype (g′) or they can first be selected for the expression of the selectable marker (with the possibility of isolating individual clones or not) and subsequently selected based on the expression of the desired phenotype (g″).

    Journal: PLoS Genetics

    Article Title: Retrovolution: HIV-Driven Evolution of Cellular Genes and Improvement of Anticancer Drug Activation

    doi: 10.1371/journal.pgen.1002904

    Figure Lengend Snippet: The Retrovolution system. Panel A . General structure of genomic RNAs for Retrovolution (top) and specific structure of the genomic RNA used in this study (bottom). Expression of the “target gene” is driven by an eukaryotic promoter (inducible or not, in this case the constitutive promoter of the human elongation factor 1-alpha, EF1-alpha); a selectable marker (here, the puromycin-resistance gene) is transcribed from a second, independent promoter (in this case the human phosphoglycerate kinase gene, hPGK) situated immediately downstream of the translation termination signal of the first gene. U3, R, U5 (LTRs) and HIV-1 cis-acting sequences are viral sequences necessary for viral genome packaging, reverse-transcription and integration. Panel B . Experimental procedure of Retrovolution. White arrows indicate the initial steps of the procedure, lilac arrows the steps involved in the generation of genetic diversity, and green arrows those involved in the selection steps. (a) HIV-based VSV-pseudotyped defective vectors are generated by transfection of producer cells (HEK-293T cells) with two plasmids leading to the production of the viral proteins (transcomplementation plasmids, pHCMV-G and pCMVΔR8.91), and one or potentially more plasmids leading to the synthesis of the genomic RNA (pSDY-dCK-Puro, outlined in panel A). The retroviral vectors generated (parental generation) are used to transduce (b) producer cells (HEK-293T) at a high MOI. Expression of the puromycin-resistance gene from proviral DNA allows the selection of the efficiently transduced cell population (c). Upon transfection of these cells with transcomplementation plasmids (d), new particles encapsidating the gRNA transcribed from the proviral DNA inserted in the cell's genome will be generated and used to transduce fresh producer cells (e). Steps (c), (d), and (e) are reiterated, producing subsequent viral generations (each cycle requires a week in the case of puromycin selection, but this time varies with the selectable marker used). To screen the library, some of the vectors are used to transduce the appropriate target cells at a low MOI (f). Transduced cells can then be directly screened on the basis of the desired phenotype (g′) or they can first be selected for the expression of the selectable marker (with the possibility of isolating individual clones or not) and subsequently selected based on the expression of the desired phenotype (g″).

    Article Snippet: Cell lines HEK-293T cells were obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco's Modified Eagle's Medium (Gibco) supplemented with 10% FBS and 100 U/ml pennicillin-100 mg/ml streptomycin.

    Techniques: Expressing, Marker, Selection, Generated, Transfection, Transduction, Clone Assay

    Screening of the dCK library. Panel A . Viability of 76 isolated clones of HEK-293T transduced with the dCK-F16 viral library at a MOI

    Journal: PLoS Genetics

    Article Title: Retrovolution: HIV-Driven Evolution of Cellular Genes and Improvement of Anticancer Drug Activation

    doi: 10.1371/journal.pgen.1002904

    Figure Lengend Snippet: Screening of the dCK library. Panel A . Viability of 76 isolated clones of HEK-293T transduced with the dCK-F16 viral library at a MOI

    Article Snippet: Cell lines HEK-293T cells were obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco's Modified Eagle's Medium (Gibco) supplemented with 10% FBS and 100 U/ml pennicillin-100 mg/ml streptomycin.

    Techniques: Isolation, Clone Assay, Transduction

    R11.1.6 specifically binds K-Ras G12D in cells, blocks K-Ras-B-Raf interaction, and inhibits signaling. ( a ) Co-localization of EGFP-R11.1.6 (green) with mApple-K-Ras G12D (red) in co-transfected HEK 293T cells. Scale bars are 10 μm. Images are representative of n = 2 biological replicates. Co-localization is quantified in Extended Data Figure 5a . ( b ) Co-immunoprecipitation of HA-tagged K-Ras G12D with cmyc-R11.1.6/YW1 in co-transfected HEK 293T cells, showing R11.1.6 specificity for K-Ras G12D. IP, immunoprecipitation; WCL, whole cell lysate. Results are representative of n = 2 biological replicates. ( c ) Co-immunoprecipitation of endogenous B-Raf with HA-tagged K-Ras G12D in co-transfected HEK 293T cells, showing inhibition of K-Ras-B-Raf binding by R11.1.6. Results are representative of n = 3 biological replicates. ( d ) Effect of R11.1.6 on phosphorylation of endogenous MEK (pMEK) and ERK (pERK) via HA-K-Ras G12D-induced signaling in co-transfected HEK 293T cells, showing inhibition of signaling by R11.1.6. Results are representative of n = 3 biological replicates. Quantification is provided in Extended Data Figure 5e . Full-length blots of the cropped ones shown here are given in Extended Data Figures 6 , 7 , and 8 .

    Journal: Scientific Reports

    Article Title: An engineered protein antagonist of K-Ras/B-Raf interaction

    doi: 10.1038/s41598-017-05889-7

    Figure Lengend Snippet: R11.1.6 specifically binds K-Ras G12D in cells, blocks K-Ras-B-Raf interaction, and inhibits signaling. ( a ) Co-localization of EGFP-R11.1.6 (green) with mApple-K-Ras G12D (red) in co-transfected HEK 293T cells. Scale bars are 10 μm. Images are representative of n = 2 biological replicates. Co-localization is quantified in Extended Data Figure 5a . ( b ) Co-immunoprecipitation of HA-tagged K-Ras G12D with cmyc-R11.1.6/YW1 in co-transfected HEK 293T cells, showing R11.1.6 specificity for K-Ras G12D. IP, immunoprecipitation; WCL, whole cell lysate. Results are representative of n = 2 biological replicates. ( c ) Co-immunoprecipitation of endogenous B-Raf with HA-tagged K-Ras G12D in co-transfected HEK 293T cells, showing inhibition of K-Ras-B-Raf binding by R11.1.6. Results are representative of n = 3 biological replicates. ( d ) Effect of R11.1.6 on phosphorylation of endogenous MEK (pMEK) and ERK (pERK) via HA-K-Ras G12D-induced signaling in co-transfected HEK 293T cells, showing inhibition of signaling by R11.1.6. Results are representative of n = 3 biological replicates. Quantification is provided in Extended Data Figure 5e . Full-length blots of the cropped ones shown here are given in Extended Data Figures 6 , 7 , and 8 .

    Article Snippet: Cell culture HEK 293T cells (ATCC) were cultured in DMEM with 10% FBS.

    Techniques: Transfection, Immunoprecipitation, Inhibition, Binding Assay

    TEM images showed well-preserved ultrastructures in the CCM-processed HEK 293T cells. In the enlarged view of the boxed region (bottom panel), smooth nuclear membranes were observed. Scale bars: 1 µm for the top panel, 500 nm for the bottom panel. Pixel resolution (x,y): 6.01 nm.

    Journal: eLife

    Article Title: High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

    doi: 10.7554/eLife.35524

    Figure Lengend Snippet: TEM images showed well-preserved ultrastructures in the CCM-processed HEK 293T cells. In the enlarged view of the boxed region (bottom panel), smooth nuclear membranes were observed. Scale bars: 1 µm for the top panel, 500 nm for the bottom panel. Pixel resolution (x,y): 6.01 nm.

    Article Snippet: Cultured cells preparation HEK 293T cells (ATCC, Gaithersburg, MD) were grown on 1.2 mm diameter punches of Aclar (two mil thick; Electron Microscopy Sciences, Hatfield, PA) for 48 hr, in a humidified cell culture incubator with 5% CO2 at 37°C.

    Techniques: Transmission Electron Microscopy

    Mutant c.2587G > A PLXNA1 activates RhoGTPase and p44/p42 MAPK signaling. A) Increased activation of RhoGTPase signaling effectors cofilin and LIMK in 293T and pancreas cancer cells expressing mutant c.2587G > A PLXNA1 (293T OE WT , 293T cells overexpressing wild type PLXNA1; 293T OE MUT , 293T cells overexpressing mutant); B) Pharmacological inhibition with the CDC42-specific small molecule inhibitor ZCL278 abrogates invasion and migration in 293T and Panc1 cells overexpressing mutant c.2587G > A, but not wild type PLXNA1, as well as SB.06 cells harboring mutant c.2587G > A PLXNA1; C) Increased actin filaments and cytoplasmatic expansion in Panc1 cells transfected with mutant PLXNA1 versus PLXNA1 wild type; D) Overexpression of c.2587G > A PLXNA1 increases p-Erk levels in 293T and pancreas cancer cells compared to wild type PLXNA1. Immunoblot of 293T and Panc1 cells probed with Erk and actin antibodies; E) Expression of the epithelial-to-mesenchymal transition markers E-cadherin, vimentin, and metalloproteinase MMP-9 analyzed by qRT-PCR (normalized ΔCt values are shown). 293T cells overexpressing wild type PLXNA1 or mutant (MT) c.2587G > A PLXNA1 were treated with 150ng/ml SEMA3A (+) or vehicle (-) for 10 minutes prior to RNA harvest and qRT-PCR analysis.

    Journal: PLoS ONE

    Article Title: Whole Genome Sequencing of Newly Established Pancreatic Cancer Lines Identifies Novel Somatic Mutation (c.2587G>A) in Axon Guidance Receptor Plexin A1 as Enhancer of Proliferation and Invasion

    doi: 10.1371/journal.pone.0149833

    Figure Lengend Snippet: Mutant c.2587G > A PLXNA1 activates RhoGTPase and p44/p42 MAPK signaling. A) Increased activation of RhoGTPase signaling effectors cofilin and LIMK in 293T and pancreas cancer cells expressing mutant c.2587G > A PLXNA1 (293T OE WT , 293T cells overexpressing wild type PLXNA1; 293T OE MUT , 293T cells overexpressing mutant); B) Pharmacological inhibition with the CDC42-specific small molecule inhibitor ZCL278 abrogates invasion and migration in 293T and Panc1 cells overexpressing mutant c.2587G > A, but not wild type PLXNA1, as well as SB.06 cells harboring mutant c.2587G > A PLXNA1; C) Increased actin filaments and cytoplasmatic expansion in Panc1 cells transfected with mutant PLXNA1 versus PLXNA1 wild type; D) Overexpression of c.2587G > A PLXNA1 increases p-Erk levels in 293T and pancreas cancer cells compared to wild type PLXNA1. Immunoblot of 293T and Panc1 cells probed with Erk and actin antibodies; E) Expression of the epithelial-to-mesenchymal transition markers E-cadherin, vimentin, and metalloproteinase MMP-9 analyzed by qRT-PCR (normalized ΔCt values are shown). 293T cells overexpressing wild type PLXNA1 or mutant (MT) c.2587G > A PLXNA1 were treated with 150ng/ml SEMA3A (+) or vehicle (-) for 10 minutes prior to RNA harvest and qRT-PCR analysis.

    Article Snippet: Transient expression and proliferation and invasion assays 293T cells were purchased from ATCC and transfected with GeneCarrierI (Epoch Life Science, Sugar Land, TX, USA) at a 4:1 (μl/μg) ratio with DNA using 3 to 5μg of plasmid DNA.

    Techniques: Mutagenesis, Activation Assay, Expressing, Inhibition, Migration, Transfection, Over Expression, Quantitative RT-PCR

    Mutant PLXNA1 mediates invasion of 293T following transfection. Invasion of 293T cells transfected with A) no plasmid, B) WT plasmid and C) mutated PLXNA1 c.2587G > A plasmid, 48 hours after administration of 150 ng/mL semaphorin 3A. *p

    Journal: PLoS ONE

    Article Title: Whole Genome Sequencing of Newly Established Pancreatic Cancer Lines Identifies Novel Somatic Mutation (c.2587G>A) in Axon Guidance Receptor Plexin A1 as Enhancer of Proliferation and Invasion

    doi: 10.1371/journal.pone.0149833

    Figure Lengend Snippet: Mutant PLXNA1 mediates invasion of 293T following transfection. Invasion of 293T cells transfected with A) no plasmid, B) WT plasmid and C) mutated PLXNA1 c.2587G > A plasmid, 48 hours after administration of 150 ng/mL semaphorin 3A. *p

    Article Snippet: Transient expression and proliferation and invasion assays 293T cells were purchased from ATCC and transfected with GeneCarrierI (Epoch Life Science, Sugar Land, TX, USA) at a 4:1 (μl/μg) ratio with DNA using 3 to 5μg of plasmid DNA.

    Techniques: Mutagenesis, Transfection, Plasmid Preparation

    Pcid2 deficiency causes H2A.Z deposition to lymphoid fate regulator genes in MPPs. a Sorted MPPs were lysed for ChIP assays. Indicated promoters were examined by qPCR. Signals were normalized to input DNA. Fold enrichment was calculated comparing with negative control (Non-pro locus). Results are shown as means ± S.D. b , c Indicated MPPs were lysed for ChIP assays with anti-H2A b or anti-SRCAP c antibody as in a . d MPP lysates were incubated with anti-SRCAP antibody. SRCAP-associated PU.1 and GATA1 were detected by immunoblotting. IP immunoprecipitation, RIgG rabbit IgG. e Indicated MPPs were lysed for ChIP assays with anti-PU.1 antibody as in a . f Expression levels of lymphoid fate regulator genes and myeloid fate regulator genes were assessed in sorted MPPs by quantitative RT-PCR. g Nuclei of MPPs were extracted for DNase I digestion assay. Chromatin accessibility were quantitated by qPCR. h Flag-PU.1, pTK and pGL3- Il7r promoter or pGL3- Ikzf1 promoter, together with the indicated shRNAs, were transfected into 293T cells for luciferase assays. Results are shown as means ± S.D. i Pcid2 and Spi1 DKO were generated as described in Methods section. Bone marrow mRNA levels of Pcid2 and Spi1 were measured by qPCR. j In vitro lymphoid differentiation assays of the indicated LMPPs were conducted as in Fig. 2c . For Pcid2 or PU.1 restoration, 1 × 10 3 HSCs were isolated from the BM of Spi1 −/− or Pcid2 −/− mice and infected with pMY-GFP-Pcid2 or pMY-GFP-PU.1 containing retrovirus and transplanted into lethally irradiated recipient mice together with 5 × 10 6 helper cells. k Cells were sorted, infected and cultured as in j and used Flt3 ligand alone thereafter. l B-cell and myeloid differentiation assays of the indicated MPPs in vitro were conducted as in Fig. 2e . m Indicated HSCs as described in j were sorted, infected and transplanted for 16 weeks. m Percentages of MPPs, n CLPs and o CMPs from the BM of indicated mice were analysed by flow cytometry. Results are shown as means ± S.D. n = 6 for each group. ** P

    Journal: Nature Communications

    Article Title: Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2

    doi: 10.1038/s41467-017-01788-7

    Figure Lengend Snippet: Pcid2 deficiency causes H2A.Z deposition to lymphoid fate regulator genes in MPPs. a Sorted MPPs were lysed for ChIP assays. Indicated promoters were examined by qPCR. Signals were normalized to input DNA. Fold enrichment was calculated comparing with negative control (Non-pro locus). Results are shown as means ± S.D. b , c Indicated MPPs were lysed for ChIP assays with anti-H2A b or anti-SRCAP c antibody as in a . d MPP lysates were incubated with anti-SRCAP antibody. SRCAP-associated PU.1 and GATA1 were detected by immunoblotting. IP immunoprecipitation, RIgG rabbit IgG. e Indicated MPPs were lysed for ChIP assays with anti-PU.1 antibody as in a . f Expression levels of lymphoid fate regulator genes and myeloid fate regulator genes were assessed in sorted MPPs by quantitative RT-PCR. g Nuclei of MPPs were extracted for DNase I digestion assay. Chromatin accessibility were quantitated by qPCR. h Flag-PU.1, pTK and pGL3- Il7r promoter or pGL3- Ikzf1 promoter, together with the indicated shRNAs, were transfected into 293T cells for luciferase assays. Results are shown as means ± S.D. i Pcid2 and Spi1 DKO were generated as described in Methods section. Bone marrow mRNA levels of Pcid2 and Spi1 were measured by qPCR. j In vitro lymphoid differentiation assays of the indicated LMPPs were conducted as in Fig. 2c . For Pcid2 or PU.1 restoration, 1 × 10 3 HSCs were isolated from the BM of Spi1 −/− or Pcid2 −/− mice and infected with pMY-GFP-Pcid2 or pMY-GFP-PU.1 containing retrovirus and transplanted into lethally irradiated recipient mice together with 5 × 10 6 helper cells. k Cells were sorted, infected and cultured as in j and used Flt3 ligand alone thereafter. l B-cell and myeloid differentiation assays of the indicated MPPs in vitro were conducted as in Fig. 2e . m Indicated HSCs as described in j were sorted, infected and transplanted for 16 weeks. m Percentages of MPPs, n CLPs and o CMPs from the BM of indicated mice were analysed by flow cytometry. Results are shown as means ± S.D. n = 6 for each group. ** P

    Article Snippet: Cell culture Human 293T cells (ATCC, CRL-3216) were cultured with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 mg/ml streptomycin.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Incubation, Immunoprecipitation, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Generated, In Vitro, Isolation, Mouse Assay, Infection, Irradiation, Cell Culture, Flow Cytometry, Cytometry

    Speedy increases cell division and decreases the G1 population. 293T cells were transiently transfected with empty vector or myc–Spy1. Cells were collected 24, 30, 36, and 48 h after transfection and analyzed by flow cytometry. (A) A representative FACS ® profile of cells overexpressing empty vector (mock) or myc–Spy1. (B) Graphic representation of the percentage of cells in G1 phase. Mock, black bars; myc–Spy1, gray bars. Error bars represent the SEM of four independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Human Speedy

    doi: 10.1083/jcb.200109045

    Figure Lengend Snippet: Speedy increases cell division and decreases the G1 population. 293T cells were transiently transfected with empty vector or myc–Spy1. Cells were collected 24, 30, 36, and 48 h after transfection and analyzed by flow cytometry. (A) A representative FACS ® profile of cells overexpressing empty vector (mock) or myc–Spy1. (B) Graphic representation of the percentage of cells in G1 phase. Mock, black bars; myc–Spy1, gray bars. Error bars represent the SEM of four independent experiments.

    Article Snippet: Cell culture and synchronization 293T cells are a human embryonic kidney cell line (American Type Culture Collection [ATCC]), Ntera-2 cells are a human teratocarcinoma cell line (ATCC), HeLa cells are a human epitheloid carcinoma cell line (ATCC), COS-1 cells are an SV40-transformed African green monkey kidney cell line (ATCC), and NIH3T3 cells are an embryonic, contact inhibited, NIH Swiss mouse cell line (ATCC).

    Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, FACS

    Endogenous Spy1 is required for normal cell growth. Spy1 RNA was depleted using RNA interference. (A) 293T cells were transiently transfected with siRNA directed against Spy1 (siSpy) (□) or siluc-GL2 control (siCntl) oligonucleotides (○). 24 h after transfection, triplicate plates were counted by trypan blue exclusion. The surviving cell number was graphed as an average of three plates (±SEM). (B) mRNA was isolated from remaining cells from each time point and was subjected to RT-PCR analysis. Lanes indicated with a plus have been treated with siSpy, and lanes indicated with a minus have been treated with siCntl. (C) Control 1.2% agarose formaldehyde gel demonstrating the quantity and quality of the mRNA used in B.

    Journal: The Journal of Cell Biology

    Article Title: Human Speedy

    doi: 10.1083/jcb.200109045

    Figure Lengend Snippet: Endogenous Spy1 is required for normal cell growth. Spy1 RNA was depleted using RNA interference. (A) 293T cells were transiently transfected with siRNA directed against Spy1 (siSpy) (□) or siluc-GL2 control (siCntl) oligonucleotides (○). 24 h after transfection, triplicate plates were counted by trypan blue exclusion. The surviving cell number was graphed as an average of three plates (±SEM). (B) mRNA was isolated from remaining cells from each time point and was subjected to RT-PCR analysis. Lanes indicated with a plus have been treated with siSpy, and lanes indicated with a minus have been treated with siCntl. (C) Control 1.2% agarose formaldehyde gel demonstrating the quantity and quality of the mRNA used in B.

    Article Snippet: Cell culture and synchronization 293T cells are a human embryonic kidney cell line (American Type Culture Collection [ATCC]), Ntera-2 cells are a human teratocarcinoma cell line (ATCC), HeLa cells are a human epitheloid carcinoma cell line (ATCC), COS-1 cells are an SV40-transformed African green monkey kidney cell line (ATCC), and NIH3T3 cells are an embryonic, contact inhibited, NIH Swiss mouse cell line (ATCC).

    Techniques: Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction

    Spy1-enhanced growth is dependent on cdk2 activation. (A) Growth curve of 293T cells transiently transfected with Spy1 (□); mock (⋄); Spy1 + dncdk2 (cdk2D145N) (Δ); mock + dncdk2 (X); Spy1 + 7 μM olomoucine ( ∇ ); mock + 7 μM olomoucine (○). Error bars represent the SEM between triplicate plates of one representative experiment. This experiment was repeated three times. (B) Western blot of lysates from each sample at 96 h. The blot was then probed for myc–Spy1 expression.

    Journal: The Journal of Cell Biology

    Article Title: Human Speedy

    doi: 10.1083/jcb.200109045

    Figure Lengend Snippet: Spy1-enhanced growth is dependent on cdk2 activation. (A) Growth curve of 293T cells transiently transfected with Spy1 (□); mock (⋄); Spy1 + dncdk2 (cdk2D145N) (Δ); mock + dncdk2 (X); Spy1 + 7 μM olomoucine ( ∇ ); mock + 7 μM olomoucine (○). Error bars represent the SEM between triplicate plates of one representative experiment. This experiment was repeated three times. (B) Western blot of lysates from each sample at 96 h. The blot was then probed for myc–Spy1 expression.

    Article Snippet: Cell culture and synchronization 293T cells are a human embryonic kidney cell line (American Type Culture Collection [ATCC]), Ntera-2 cells are a human teratocarcinoma cell line (ATCC), HeLa cells are a human epitheloid carcinoma cell line (ATCC), COS-1 cells are an SV40-transformed African green monkey kidney cell line (ATCC), and NIH3T3 cells are an embryonic, contact inhibited, NIH Swiss mouse cell line (ATCC).

    Techniques: Activation Assay, Transfection, Western Blot, Expressing

    Human Spy1 mRNA is present in a variety of human tissues and immortalized cell lines. (A) RT-PCR of mRNA from 15 different human tissues. Lane 1 is a negative RT-PCR control containing no mRNA. Lane 2 is a positive RT-PCR control using control RNA with control primers. Human tissue samples are as follows: lane 3, thymus; 4, salivary gland; 5, liver; 6, fetal brain; 7, adrenal gland; 8, bone marrow; 9, fetal liver; 10, lung; 11, skeletal muscle; 12, thyroid; 13, brain/cerebellum; 14, heart; 15, placenta; 16, spleen; 17, trachea. (B) Control formaldehyde gel demonstrating the quality and quantity of the mRNA used in A. (C) RT-PCR of mRNA prepared from Ntera-2 cells (lane 3), 293T cells (lane 4), Spy1-transfected 293T cells (lane 5), and HeLa cells (lane 6). Lanes 1 and 2 are RT-PCR controls with a control mRNA and no mRNA, respectively. (D) Control formaldehyde gel demonstrating the quality and quantity of the mRNA used in C. Lanes 1–4 correspond to lanes 3–6 in C. (E) RT-PCR of mRNA prepared from synchronized 293T cells at various stages of the cell cycle. Lanes 1 and 2 are RT-PCR controls with no mRNA and control mRNA, respectively. (F) Control formaldehyde gel demonstrating the quantity and quality of the mRNA used in E. Lanes 1–5 correspond to lanes 3–7 in E.

    Journal: The Journal of Cell Biology

    Article Title: Human Speedy

    doi: 10.1083/jcb.200109045

    Figure Lengend Snippet: Human Spy1 mRNA is present in a variety of human tissues and immortalized cell lines. (A) RT-PCR of mRNA from 15 different human tissues. Lane 1 is a negative RT-PCR control containing no mRNA. Lane 2 is a positive RT-PCR control using control RNA with control primers. Human tissue samples are as follows: lane 3, thymus; 4, salivary gland; 5, liver; 6, fetal brain; 7, adrenal gland; 8, bone marrow; 9, fetal liver; 10, lung; 11, skeletal muscle; 12, thyroid; 13, brain/cerebellum; 14, heart; 15, placenta; 16, spleen; 17, trachea. (B) Control formaldehyde gel demonstrating the quality and quantity of the mRNA used in A. (C) RT-PCR of mRNA prepared from Ntera-2 cells (lane 3), 293T cells (lane 4), Spy1-transfected 293T cells (lane 5), and HeLa cells (lane 6). Lanes 1 and 2 are RT-PCR controls with a control mRNA and no mRNA, respectively. (D) Control formaldehyde gel demonstrating the quality and quantity of the mRNA used in C. Lanes 1–4 correspond to lanes 3–6 in C. (E) RT-PCR of mRNA prepared from synchronized 293T cells at various stages of the cell cycle. Lanes 1 and 2 are RT-PCR controls with no mRNA and control mRNA, respectively. (F) Control formaldehyde gel demonstrating the quantity and quality of the mRNA used in E. Lanes 1–5 correspond to lanes 3–7 in E.

    Article Snippet: Cell culture and synchronization 293T cells are a human embryonic kidney cell line (American Type Culture Collection [ATCC]), Ntera-2 cells are a human teratocarcinoma cell line (ATCC), HeLa cells are a human epitheloid carcinoma cell line (ATCC), COS-1 cells are an SV40-transformed African green monkey kidney cell line (ATCC), and NIH3T3 cells are an embryonic, contact inhibited, NIH Swiss mouse cell line (ATCC).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection

    Human Spy1 binds to and activates cdk2. (A) Indirect immunofluorescence on COS-1 cells transiently transfected with human myc-tagged Spy1 (myc–Spy1; bottom left) or with empty vector (mock; top left) shows myc–Spy1 in the nucleus. The corresponding panels on the right are stained with Hoechst dye to illuminate the nuclei of all the cells present. (B) Coimmunoprecipitation of transiently transfected myc–Spy1 with endogenous cdk2 from 293T cells. Western blot analysis using antibodies against the myc epitope tag (top) or against cdk2 (bottom). Lane 1 is lysate from mock-transfected cells. Lane 2 is lysate from myc–Spy1-transfected cells. Lanes 3 and 4 are immunoprecipitations (IPs) from mock-transfected cells, and lanes 5 and 6 are immunoprecipitations from myc–Spy1-transfected cells using either anti-cdk2 (lanes 3 and 5) or anti-myc (lanes 4 and 6). (C) Top panel shows histone H1 phosphorylation assay. 293T cells were transiently transfected with empty vector (mock) or myc–Spy1. 24 h after transfection, cells were harvested and immunoprecipitated with antibodies against cdk2. Lane 1 is histone H1 alone (no IP). Lanes 2 (mock) and 3 (myc–Spy1) are cdk2 IPs. Bottom panel shows blot of cdk2 in IPs from mock and myc–Spy1 cells. (D) Top panel shows histone H1 phosphorylation assay. 293T cells were transiently transfected with empty vector (mock) (lane 1) or myc–Spy1 (lane 2). 24 h after transfection, cells were harvested and immunoprecipitated with antibodies against cdc2. Coomassie blue staining showing equal loading of cdc2 is presented in the bottom panel.

    Journal: The Journal of Cell Biology

    Article Title: Human Speedy

    doi: 10.1083/jcb.200109045

    Figure Lengend Snippet: Human Spy1 binds to and activates cdk2. (A) Indirect immunofluorescence on COS-1 cells transiently transfected with human myc-tagged Spy1 (myc–Spy1; bottom left) or with empty vector (mock; top left) shows myc–Spy1 in the nucleus. The corresponding panels on the right are stained with Hoechst dye to illuminate the nuclei of all the cells present. (B) Coimmunoprecipitation of transiently transfected myc–Spy1 with endogenous cdk2 from 293T cells. Western blot analysis using antibodies against the myc epitope tag (top) or against cdk2 (bottom). Lane 1 is lysate from mock-transfected cells. Lane 2 is lysate from myc–Spy1-transfected cells. Lanes 3 and 4 are immunoprecipitations (IPs) from mock-transfected cells, and lanes 5 and 6 are immunoprecipitations from myc–Spy1-transfected cells using either anti-cdk2 (lanes 3 and 5) or anti-myc (lanes 4 and 6). (C) Top panel shows histone H1 phosphorylation assay. 293T cells were transiently transfected with empty vector (mock) or myc–Spy1. 24 h after transfection, cells were harvested and immunoprecipitated with antibodies against cdk2. Lane 1 is histone H1 alone (no IP). Lanes 2 (mock) and 3 (myc–Spy1) are cdk2 IPs. Bottom panel shows blot of cdk2 in IPs from mock and myc–Spy1 cells. (D) Top panel shows histone H1 phosphorylation assay. 293T cells were transiently transfected with empty vector (mock) (lane 1) or myc–Spy1 (lane 2). 24 h after transfection, cells were harvested and immunoprecipitated with antibodies against cdc2. Coomassie blue staining showing equal loading of cdc2 is presented in the bottom panel.

    Article Snippet: Cell culture and synchronization 293T cells are a human embryonic kidney cell line (American Type Culture Collection [ATCC]), Ntera-2 cells are a human teratocarcinoma cell line (ATCC), HeLa cells are a human epitheloid carcinoma cell line (ATCC), COS-1 cells are an SV40-transformed African green monkey kidney cell line (ATCC), and NIH3T3 cells are an embryonic, contact inhibited, NIH Swiss mouse cell line (ATCC).

    Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Staining, Western Blot, Phosphorylation Assay, Immunoprecipitation

    Expression of Spy1 increases both the rate of cell replication and division. (A) Growth curve of 293T cells transiently transfected with myc–Spy1 (□) or empty vector (○). Cells were collected at the indicated times, counted on a Coulter counter, and replated. The graph is the average of three independent experiments. Error bars were calculated using SEM. (B) Increase of BrdU incorporation in myc–Spy1-transfected 293T cells. Cells were harvested at day 2, stained for BrdU, and the positive cells were counted via fluorescence microscopy. Error bars represent the standard error of three experiments. (C) MTT analysis of 293T cells expressing myc–Spy1 (□) or empty vector (○). Cells were collected at the indicated times, treated, and then the absorbance was taken at 570 nm to determine the relative levels of MTT. Bars represent the standard error between four separate transfections within one representative experiment. (D) Histone H3 phosphorylation in myc–Spy1-transfected 293T cells. Cells were harvested at day 2, stained for phosphorylated histone H3, and the positive cells were counted via fluorescence microscopy. Bars represent the standard error of three separate experiments.

    Journal: The Journal of Cell Biology

    Article Title: Human Speedy

    doi: 10.1083/jcb.200109045

    Figure Lengend Snippet: Expression of Spy1 increases both the rate of cell replication and division. (A) Growth curve of 293T cells transiently transfected with myc–Spy1 (□) or empty vector (○). Cells were collected at the indicated times, counted on a Coulter counter, and replated. The graph is the average of three independent experiments. Error bars were calculated using SEM. (B) Increase of BrdU incorporation in myc–Spy1-transfected 293T cells. Cells were harvested at day 2, stained for BrdU, and the positive cells were counted via fluorescence microscopy. Error bars represent the standard error of three experiments. (C) MTT analysis of 293T cells expressing myc–Spy1 (□) or empty vector (○). Cells were collected at the indicated times, treated, and then the absorbance was taken at 570 nm to determine the relative levels of MTT. Bars represent the standard error between four separate transfections within one representative experiment. (D) Histone H3 phosphorylation in myc–Spy1-transfected 293T cells. Cells were harvested at day 2, stained for phosphorylated histone H3, and the positive cells were counted via fluorescence microscopy. Bars represent the standard error of three separate experiments.

    Article Snippet: Cell culture and synchronization 293T cells are a human embryonic kidney cell line (American Type Culture Collection [ATCC]), Ntera-2 cells are a human teratocarcinoma cell line (ATCC), HeLa cells are a human epitheloid carcinoma cell line (ATCC), COS-1 cells are an SV40-transformed African green monkey kidney cell line (ATCC), and NIH3T3 cells are an embryonic, contact inhibited, NIH Swiss mouse cell line (ATCC).

    Techniques: Expressing, Transfection, Plasmid Preparation, BrdU Incorporation Assay, Staining, Fluorescence, Microscopy, MTT Assay

    An N-terminal fragment corresponding to residues 175–244 of SOCS5 can directly bind JAK1. (A) SPR analysis of SOCS5 175–244 fragment binding to the JAK JH1 domain. Serially diluted JAK JH1 domains (62.5 nM–2 μM) were flowed over immobilised SOCS5 175–244 protein. Upper panels represent sensorgrams showing the kinetics of binding. Lower panels show steady-state analysis. (B) 293T cells were transfected with the Stat6 reporter and increasing amounts of cDNA expressing Flag-tagged SOCS5 (3.13–100 ng) or SOCS5 lacking the conserved N-terminal fragment (9.5–300 ng; Δ175–244) and stimulated overnight with 10 ng/mL rhIL-4. Cells were lysed and induced luciferase activity measured and normalised according to Renilla activity. Data are expressed as arbitrary units and represent the mean of triplicates ± SD. Cell lysates were analyzed by Western blotting for Flag-tagged proteins (SOCS5 upper; Δ175–244 lower panel); images were generated from the same gel and exposure. (C) Recombinant SOCS5 JIR or SOCS3 was incubated with 20 nM JAK1 and GST-JAK2 activation peptide (substrate; GST-J) for 15 min in the presence of 2.5 mM Mg/ 32 P-γ-ATP at 37°C. Incorporation of 32 P was visualised by autoradiography (top panel) and protein input by SDS-PAGE and Coomassie staining (lower panel).

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

    doi: 10.1371/journal.pone.0070536

    Figure Lengend Snippet: An N-terminal fragment corresponding to residues 175–244 of SOCS5 can directly bind JAK1. (A) SPR analysis of SOCS5 175–244 fragment binding to the JAK JH1 domain. Serially diluted JAK JH1 domains (62.5 nM–2 μM) were flowed over immobilised SOCS5 175–244 protein. Upper panels represent sensorgrams showing the kinetics of binding. Lower panels show steady-state analysis. (B) 293T cells were transfected with the Stat6 reporter and increasing amounts of cDNA expressing Flag-tagged SOCS5 (3.13–100 ng) or SOCS5 lacking the conserved N-terminal fragment (9.5–300 ng; Δ175–244) and stimulated overnight with 10 ng/mL rhIL-4. Cells were lysed and induced luciferase activity measured and normalised according to Renilla activity. Data are expressed as arbitrary units and represent the mean of triplicates ± SD. Cell lysates were analyzed by Western blotting for Flag-tagged proteins (SOCS5 upper; Δ175–244 lower panel); images were generated from the same gel and exposure. (C) Recombinant SOCS5 JIR or SOCS3 was incubated with 20 nM JAK1 and GST-JAK2 activation peptide (substrate; GST-J) for 15 min in the presence of 2.5 mM Mg/ 32 P-γ-ATP at 37°C. Incorporation of 32 P was visualised by autoradiography (top panel) and protein input by SDS-PAGE and Coomassie staining (lower panel).

    Article Snippet: Transient transfection of 293T cells 293T cells were maintained in DMEM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% fetal bovine serum (Sigma).

    Techniques: SPR Assay, Binding Assay, Transfection, Expressing, Luciferase, Activity Assay, Western Blot, Generated, Recombinant, Incubation, Activation Assay, Autoradiography, SDS Page, Staining

    SOCS5-SH2 domain binding analysis and identification of Shc-1 pY317 as a high affinity-potential binding target. SPR analysis of phosphopeptide binding to the SOCS5-SH2 domain. A constant amount of recombinant SOCS5 was mixed with serially diluted phosphopeptides (0.4–10 µM) and flowed over immobilised Shc-1 pY317 peptide. The response units are expressed as a percentage of maximal binding in the absence of competitor and are plotted against the concentration of competitor peptide. Steady-state analysis at saturation of binding was used to derive the K D values for the respective phosphopeptides. Binding analysis of ( A ) JAK, Shc-1, or wild-type and ( B ) mutated EGF-R phosphopeptides. Phosphopeptide sequences and the respective K D values are shown in the right-hand side table. Yellow boxes highlight residues replaced by an alanine residue. ( C ) Structural model of the SOCS5-SH2-Shc-1 peptide complex. A homology model for the SOCS5-SH2 domain was built using the SOCS4 crystal structure as a template (PDB code 2IZV). The Shc-1 pY317 peptide was modelled from the SOCS3-gp130 crystal structure (PDB code 2HMH). Side chains were optimized using ICM-PRO (Molsoft). The backbone of the flexible EF and BG loops was fixed in the apo-SOCS4 conformation, but is likely to adjust on peptide binding to maximize interactions. Predicted hydrogen bonds are shown as dashed lines. ( D ) SOCS5 interacts with full-length Shc-1 protein. 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence (+) or absence of cDNA encoding Flag-tagged Shc-1 or alternatively, with cDNA encoding Flag-tagged SOCS5 alone. Cells were treated with 10 μM MG132 for 3.5 h prior to treatment with sodium pervanadate solution for 30 min. Cells were then lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (αSOCS5). The blots were stripped and reprobed with a phospho-specific antibody for Shc-1-Y317 (middle panel). Cell lysates were analyzed by Western blot with anti-SOCS5 (lower panel).

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

    doi: 10.1371/journal.pone.0070536

    Figure Lengend Snippet: SOCS5-SH2 domain binding analysis and identification of Shc-1 pY317 as a high affinity-potential binding target. SPR analysis of phosphopeptide binding to the SOCS5-SH2 domain. A constant amount of recombinant SOCS5 was mixed with serially diluted phosphopeptides (0.4–10 µM) and flowed over immobilised Shc-1 pY317 peptide. The response units are expressed as a percentage of maximal binding in the absence of competitor and are plotted against the concentration of competitor peptide. Steady-state analysis at saturation of binding was used to derive the K D values for the respective phosphopeptides. Binding analysis of ( A ) JAK, Shc-1, or wild-type and ( B ) mutated EGF-R phosphopeptides. Phosphopeptide sequences and the respective K D values are shown in the right-hand side table. Yellow boxes highlight residues replaced by an alanine residue. ( C ) Structural model of the SOCS5-SH2-Shc-1 peptide complex. A homology model for the SOCS5-SH2 domain was built using the SOCS4 crystal structure as a template (PDB code 2IZV). The Shc-1 pY317 peptide was modelled from the SOCS3-gp130 crystal structure (PDB code 2HMH). Side chains were optimized using ICM-PRO (Molsoft). The backbone of the flexible EF and BG loops was fixed in the apo-SOCS4 conformation, but is likely to adjust on peptide binding to maximize interactions. Predicted hydrogen bonds are shown as dashed lines. ( D ) SOCS5 interacts with full-length Shc-1 protein. 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence (+) or absence of cDNA encoding Flag-tagged Shc-1 or alternatively, with cDNA encoding Flag-tagged SOCS5 alone. Cells were treated with 10 μM MG132 for 3.5 h prior to treatment with sodium pervanadate solution for 30 min. Cells were then lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (αSOCS5). The blots were stripped and reprobed with a phospho-specific antibody for Shc-1-Y317 (middle panel). Cell lysates were analyzed by Western blot with anti-SOCS5 (lower panel).

    Article Snippet: Transient transfection of 293T cells 293T cells were maintained in DMEM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% fetal bovine serum (Sigma).

    Techniques: Binding Assay, SPR Assay, Recombinant, Concentration Assay, Transfection, Western Blot

    SOCS5 can specifically block JAK1 and JAK2 autophosphorylation and the SOCS5 N-terminus is critical for inhibition of JAK. (A) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS1-SOCS7. 293T cells were transfected with cDNA encoding (B) Flag-tagged JAK2, (C) JAK3 or TYK2 in the presence of either SOCS1 or SOCS5. (D E) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS5 or various SOCS5 mutants with either N-terminal truncations (Δ369, Δ349, Δ313, Δ171, Δ110) or with His360 (H360A), the SH2 domain (mSH2) or SOCS box (mSB) mutated. (A–E) Cells were lysed and anti-Flag immunoprecipitates analyzed by Western blot with phospho-specific (JAK1: A, D E; JAK2: B) or anti-phosphotyrosine antibodies (αPY) (JAK1, JAK3 TYK2; C) (upper panels). The blots were stripped and reprobed with rat anti-Flag antibody (lower panels). (F) 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence or absence of cDNA encoding Flag-tagged JAK1, JAK2, JAK3 or TYK2. Cells were lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (top panel). The blot was stripped and reprobed with anti-Flag antibodies (middle panel). Cell lysates were blotted with anti-SOCS5 antibodies (bottom panel). Panels A, B, D and E are 10% acrylamide gels. Panels C and F are 4–12% gradient gels.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

    doi: 10.1371/journal.pone.0070536

    Figure Lengend Snippet: SOCS5 can specifically block JAK1 and JAK2 autophosphorylation and the SOCS5 N-terminus is critical for inhibition of JAK. (A) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS1-SOCS7. 293T cells were transfected with cDNA encoding (B) Flag-tagged JAK2, (C) JAK3 or TYK2 in the presence of either SOCS1 or SOCS5. (D E) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS5 or various SOCS5 mutants with either N-terminal truncations (Δ369, Δ349, Δ313, Δ171, Δ110) or with His360 (H360A), the SH2 domain (mSH2) or SOCS box (mSB) mutated. (A–E) Cells were lysed and anti-Flag immunoprecipitates analyzed by Western blot with phospho-specific (JAK1: A, D E; JAK2: B) or anti-phosphotyrosine antibodies (αPY) (JAK1, JAK3 TYK2; C) (upper panels). The blots were stripped and reprobed with rat anti-Flag antibody (lower panels). (F) 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence or absence of cDNA encoding Flag-tagged JAK1, JAK2, JAK3 or TYK2. Cells were lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (top panel). The blot was stripped and reprobed with anti-Flag antibodies (middle panel). Cell lysates were blotted with anti-SOCS5 antibodies (bottom panel). Panels A, B, D and E are 10% acrylamide gels. Panels C and F are 4–12% gradient gels.

    Article Snippet: Transient transfection of 293T cells 293T cells were maintained in DMEM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% fetal bovine serum (Sigma).

    Techniques: Blocking Assay, Inhibition, Transfection, Western Blot

    SOCS5 inhibits JAK1 kinase activity. (A) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS1 or SOCS5. Anti-Flag immunoprecipitates were incubated in the presence of 32 P-γ-ATP at 37°C. Incorporation of 32 P was visualised by autoradiography (top panel). Immunoprecipitates were analyzed by Western blot with anti-Flag antibodies (lower panel). (B) cDNAs encoding Flag-tagged SOCS1, SOCS3, SOCS5 or JAK1 were independently transfected into 293T cells. Proteins were immunoprecipitated using anti-Flag antibody, and eluted from the resin by competition with Flag peptide. Proteins were then mixed and an in vitro kinase assay performed. JAK1 autophosphorylation and phosphorylation of the GST-Jak2 activation peptide (substrate; GST-J) (top panel) were assessed by Western blotting with phospho-specific antibodies. A sample of the reaction mix was analyzed by Coomassie staining to show substrate input (lower panel).

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

    doi: 10.1371/journal.pone.0070536

    Figure Lengend Snippet: SOCS5 inhibits JAK1 kinase activity. (A) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS1 or SOCS5. Anti-Flag immunoprecipitates were incubated in the presence of 32 P-γ-ATP at 37°C. Incorporation of 32 P was visualised by autoradiography (top panel). Immunoprecipitates were analyzed by Western blot with anti-Flag antibodies (lower panel). (B) cDNAs encoding Flag-tagged SOCS1, SOCS3, SOCS5 or JAK1 were independently transfected into 293T cells. Proteins were immunoprecipitated using anti-Flag antibody, and eluted from the resin by competition with Flag peptide. Proteins were then mixed and an in vitro kinase assay performed. JAK1 autophosphorylation and phosphorylation of the GST-Jak2 activation peptide (substrate; GST-J) (top panel) were assessed by Western blotting with phospho-specific antibodies. A sample of the reaction mix was analyzed by Coomassie staining to show substrate input (lower panel).

    Article Snippet: Transient transfection of 293T cells 293T cells were maintained in DMEM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% fetal bovine serum (Sigma).

    Techniques: Activity Assay, Transfection, Incubation, Autoradiography, Western Blot, Immunoprecipitation, In Vitro, Kinase Assay, Activation Assay, Staining

    Influence of NiV-N on the STAT nuclear import system. (A) The associations between STAT1 and Impα5, Impα6, and Impα7 in the presence of N protein were evaluated by immunoprecipitation (IP). The myc-tagged importins were precipitated with an anti-myc antibody, and the coprecipitation of pSTAT1 with Impα5, Impα6, or Impα7 was evaluated in the presence or absence of N protein. A myc-tagged Impα1 construct was employed as a negative control. P protein served as a positive-control antagonist of the interaction between Impα5 and STAT1. (B) The interaction between NiV-N and HA-Impβ1 or HA-Ran was investigated with immunoprecipitation assays using anti-HA and anti-NiV-N antibodies. The myc-Impα5, myc-Impβ1, and NiV-P proteins were included as positive controls for immunoprecipitation. (C) 293T cells were transfected with pCAGGS-NiV-N (encoding a C-terminal HA tag). After 24 h, the cells were treated with 1,000 U/ml IFN-α for 30 min, and immunoprecipitation was conducted using anti-HA antibody. STAT1, STAT2, STAT3, and N protein in the lysates were detected with anti-STAT1 (E-23), -2 (C-20), and -3 (H-190) and anti-N protein antibodies, respectively. The P protein served as a positive control for an N-binding protein. For panels A to C, the experiments were independently repeated three times, and representative blots are displayed. (D) A reporter assay was conducted using 293T cells transfected with the NiV-N or N-S451A plasmid. The expression levels of each N protein and GAPDH are shown. Error bars indicate standard deviations. n.s., not significant. N-S451A-expressing Cos7 cells were treated with 2,000 U/ml of IFN-α. N-S451A and STAT1 were detected with specific antibodies and are shown as z-stack immunofluorescence images. Arrowheads and arrows indicate an N-S451A-expressing and a non-N-S451A-expressing cell, respectively. The experiment was independently conducted three times. (E) The expression plasmids for NiV-N and EGFP-Kir/Gem-W268G (referred to here as rKir/Gem) were transfected into Cos7 cells, and NiV-N was detected with an anti-NiV-N polyclonal antibody. The nuclei were stained with Hoechst dye. Images shown are z-stack data. The arrowheads and arrows point to a NiV-N-expressing and a non-NiV-N-expressing cell, respectively. The bar graph indicates the statistical evaluation of the rKir/Gem distribution. The scores were determined by counting approximately 60 cells from five randomly selected fields. n.s., not significant.

    Journal: Journal of Virology

    Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

    doi: 10.1128/JVI.01136-17

    Figure Lengend Snippet: Influence of NiV-N on the STAT nuclear import system. (A) The associations between STAT1 and Impα5, Impα6, and Impα7 in the presence of N protein were evaluated by immunoprecipitation (IP). The myc-tagged importins were precipitated with an anti-myc antibody, and the coprecipitation of pSTAT1 with Impα5, Impα6, or Impα7 was evaluated in the presence or absence of N protein. A myc-tagged Impα1 construct was employed as a negative control. P protein served as a positive-control antagonist of the interaction between Impα5 and STAT1. (B) The interaction between NiV-N and HA-Impβ1 or HA-Ran was investigated with immunoprecipitation assays using anti-HA and anti-NiV-N antibodies. The myc-Impα5, myc-Impβ1, and NiV-P proteins were included as positive controls for immunoprecipitation. (C) 293T cells were transfected with pCAGGS-NiV-N (encoding a C-terminal HA tag). After 24 h, the cells were treated with 1,000 U/ml IFN-α for 30 min, and immunoprecipitation was conducted using anti-HA antibody. STAT1, STAT2, STAT3, and N protein in the lysates were detected with anti-STAT1 (E-23), -2 (C-20), and -3 (H-190) and anti-N protein antibodies, respectively. The P protein served as a positive control for an N-binding protein. For panels A to C, the experiments were independently repeated three times, and representative blots are displayed. (D) A reporter assay was conducted using 293T cells transfected with the NiV-N or N-S451A plasmid. The expression levels of each N protein and GAPDH are shown. Error bars indicate standard deviations. n.s., not significant. N-S451A-expressing Cos7 cells were treated with 2,000 U/ml of IFN-α. N-S451A and STAT1 were detected with specific antibodies and are shown as z-stack immunofluorescence images. Arrowheads and arrows indicate an N-S451A-expressing and a non-N-S451A-expressing cell, respectively. The experiment was independently conducted three times. (E) The expression plasmids for NiV-N and EGFP-Kir/Gem-W268G (referred to here as rKir/Gem) were transfected into Cos7 cells, and NiV-N was detected with an anti-NiV-N polyclonal antibody. The nuclei were stained with Hoechst dye. Images shown are z-stack data. The arrowheads and arrows point to a NiV-N-expressing and a non-NiV-N-expressing cell, respectively. The bar graph indicates the statistical evaluation of the rKir/Gem distribution. The scores were determined by counting approximately 60 cells from five randomly selected fields. n.s., not significant.

    Article Snippet: HEK-293 (human embryonic kidney cells), 293T (HEK-293 cells stably expressing the simian virus 40 [SV40] large T antigen), HeLa (human epithelial carcinoma cells), and Cos7 (SV40-transformed African green monkey kidney fibroblasts) cells ( ) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 5% fetal bovine serum (JRH Bioscience), 2 mM l -glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 5% CO2 .

    Techniques: Immunoprecipitation, Construct, Negative Control, Positive Control, Transfection, Binding Assay, Reporter Assay, Plasmid Preparation, Expressing, Immunofluorescence, Staining

    Type I and II IFN responses in the presence or absence of henipavirus N protein. (A) pISRE-Luc and pGAS-Luc reporter gene assays with various concentrations of pCAGGS-NiV-N or -HeV-N plasmid (0.1, 0.3, 0.6, and 1.2 μg) after IFN-α or -γ stimulation in 293T cells. The transfection volume of DNA (pCAGGS-NiV-N [or -HeV-N] and the pCAGGS empty vector) was adjusted to a total of 1.2 μg. The phRL-TK (Int−) vector was cotransfected as a control for transfection efficiency. IFN responses are shown as relative luc activities compared to the Rluc activity from the control vector. Error bars indicate standard deviations. (B) 293T cells were transfected with reporter plasmids, phRL-TK (Int−), and pCAGGS-MV-N, -HeV-N, -NiV-N, or -NiV-P or pCMV-myc-NiV-V or -NiV-W. At 24 h posttransfection, reporter assays were conducted. Error bars indicate standard deviations. P values were calculated by two-tailed Student's t test. *, P

    Journal: Journal of Virology

    Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

    doi: 10.1128/JVI.01136-17

    Figure Lengend Snippet: Type I and II IFN responses in the presence or absence of henipavirus N protein. (A) pISRE-Luc and pGAS-Luc reporter gene assays with various concentrations of pCAGGS-NiV-N or -HeV-N plasmid (0.1, 0.3, 0.6, and 1.2 μg) after IFN-α or -γ stimulation in 293T cells. The transfection volume of DNA (pCAGGS-NiV-N [or -HeV-N] and the pCAGGS empty vector) was adjusted to a total of 1.2 μg. The phRL-TK (Int−) vector was cotransfected as a control for transfection efficiency. IFN responses are shown as relative luc activities compared to the Rluc activity from the control vector. Error bars indicate standard deviations. (B) 293T cells were transfected with reporter plasmids, phRL-TK (Int−), and pCAGGS-MV-N, -HeV-N, -NiV-N, or -NiV-P or pCMV-myc-NiV-V or -NiV-W. At 24 h posttransfection, reporter assays were conducted. Error bars indicate standard deviations. P values were calculated by two-tailed Student's t test. *, P

    Article Snippet: HEK-293 (human embryonic kidney cells), 293T (HEK-293 cells stably expressing the simian virus 40 [SV40] large T antigen), HeLa (human epithelial carcinoma cells), and Cos7 (SV40-transformed African green monkey kidney fibroblasts) cells ( ) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 5% fetal bovine serum (JRH Bioscience), 2 mM l -glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 5% CO2 .

    Techniques: Plasmid Preparation, Transfection, Activity Assay, Two Tailed Test

    Analysis of the mechanism by which NiV-N prevents STAT nuclear accumulation. (A) HeLa cells were treated with 20 nM LMB at 37°C for 1 h or 16 h, and STAT2 was detected by use of an anti-STAT2 polyclonal antibody (C-20). (B) HeLa cells were transfected with a NiV-N expression vector. At 24 h posttransfection, the cells were left untreated or treated with 20 nM LMB at 37°C for 1 h. After treatment with 1,000 U/ml IFN-α, the localization of STAT2 and N protein was investigated by use of anti-STAT2 (C-20) and anti-N protein antibodies. Arrowheads indicate cytoplasmic STAT2 in a NiV-N-expressing cell. Images shown are z-stack data. The experiment was repeated three times independently. (C) 293T cells transfected with the pGAS-Luc plasmid and the pCAGGS-NiV-N or -mP plasmid were treated with 1,000 U/ml IFN-γ, and the reporter gene activity was measured. **, P

    Journal: Journal of Virology

    Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

    doi: 10.1128/JVI.01136-17

    Figure Lengend Snippet: Analysis of the mechanism by which NiV-N prevents STAT nuclear accumulation. (A) HeLa cells were treated with 20 nM LMB at 37°C for 1 h or 16 h, and STAT2 was detected by use of an anti-STAT2 polyclonal antibody (C-20). (B) HeLa cells were transfected with a NiV-N expression vector. At 24 h posttransfection, the cells were left untreated or treated with 20 nM LMB at 37°C for 1 h. After treatment with 1,000 U/ml IFN-α, the localization of STAT2 and N protein was investigated by use of anti-STAT2 (C-20) and anti-N protein antibodies. Arrowheads indicate cytoplasmic STAT2 in a NiV-N-expressing cell. Images shown are z-stack data. The experiment was repeated three times independently. (C) 293T cells transfected with the pGAS-Luc plasmid and the pCAGGS-NiV-N or -mP plasmid were treated with 1,000 U/ml IFN-γ, and the reporter gene activity was measured. **, P

    Article Snippet: HEK-293 (human embryonic kidney cells), 293T (HEK-293 cells stably expressing the simian virus 40 [SV40] large T antigen), HeLa (human epithelial carcinoma cells), and Cos7 (SV40-transformed African green monkey kidney fibroblasts) cells ( ) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 5% fetal bovine serum (JRH Bioscience), 2 mM l -glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 5% CO2 .

    Techniques: Transfection, Expressing, Plasmid Preparation, Activity Assay

    Influence of N protein on expression of ISGs. (A) 293T cells were treated with 1,000 U/ml IFN-α in the presence or absence of NiV-N protein. The component proteins were fractionated into nuclear and cytoplasmic fractions. The whole-cell lysate and fractionated samples were subjected to SDS-PAGE to examine if the NiV-N protein decreased the nuclear pSTAT1 level. The relative nuclear STAT1 level (pSTAT1/histone H3) was measured by densitometric analysis using ImageJ software, and mean values for three independent experiments are shown. Error bars indicate standard deviations. (B) 293T cells were transfected with a NiV-N expression plasmid or empty vector, and a chromatin immunoprecipitation assay was carried out with anti-STAT1 antibody (E-23) or control IgG (ab37415-5) after treatment with 1,000 U/ml IFN-α. Enrichment of ISG promoters (IFIT2, MX2, OAS1, and PKR) was measured by quantitative PCR, and the amount of precipitated DNA relative to the amount of input DNA is shown as a percentage of the input. The experiment was repeated three times independently, and error bars show standard deviations. *, P

    Journal: Journal of Virology

    Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

    doi: 10.1128/JVI.01136-17

    Figure Lengend Snippet: Influence of N protein on expression of ISGs. (A) 293T cells were treated with 1,000 U/ml IFN-α in the presence or absence of NiV-N protein. The component proteins were fractionated into nuclear and cytoplasmic fractions. The whole-cell lysate and fractionated samples were subjected to SDS-PAGE to examine if the NiV-N protein decreased the nuclear pSTAT1 level. The relative nuclear STAT1 level (pSTAT1/histone H3) was measured by densitometric analysis using ImageJ software, and mean values for three independent experiments are shown. Error bars indicate standard deviations. (B) 293T cells were transfected with a NiV-N expression plasmid or empty vector, and a chromatin immunoprecipitation assay was carried out with anti-STAT1 antibody (E-23) or control IgG (ab37415-5) after treatment with 1,000 U/ml IFN-α. Enrichment of ISG promoters (IFIT2, MX2, OAS1, and PKR) was measured by quantitative PCR, and the amount of precipitated DNA relative to the amount of input DNA is shown as a percentage of the input. The experiment was repeated three times independently, and error bars show standard deviations. *, P

    Article Snippet: HEK-293 (human embryonic kidney cells), 293T (HEK-293 cells stably expressing the simian virus 40 [SV40] large T antigen), HeLa (human epithelial carcinoma cells), and Cos7 (SV40-transformed African green monkey kidney fibroblasts) cells ( ) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 5% fetal bovine serum (JRH Bioscience), 2 mM l -glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 5% CO2 .

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

    NDRG2 is a novel PTEN-interacting protein. ( a ) The KK1 cell lysates transfected with the mock or FLAG-NDRG2 vector were immunoprecipitated with an anti-FLAG antibody, and the western blots were probed with the indicated antibodies. IP, immunoprecipitation. The data are representative of three experiments. ( b ) Exogenously expressed PTEN and NDRG2 were co-immunoprecipitated in 293T cells (WB, western blot). The data are representative of three experiments. ( c ) The co-immunoprecipitation of endogenous PTEN and NDRG2 was performed in MOLT4 cell lysates. On the input lane (−), 1/200 of the input was loaded for detection of NDRG2. Asterisk, nonspecific band. The data are representative of three experiments. ( d ) The co-localization of endogenous PTEN and NDRG2 was determined in MOLT4 cells. The nuclei were labelled with DAPI. Scale bar, 10 μm. The data are representative of three experiments.

    Journal: Nature Communications

    Article Title: Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers

    doi: 10.1038/ncomms4393

    Figure Lengend Snippet: NDRG2 is a novel PTEN-interacting protein. ( a ) The KK1 cell lysates transfected with the mock or FLAG-NDRG2 vector were immunoprecipitated with an anti-FLAG antibody, and the western blots were probed with the indicated antibodies. IP, immunoprecipitation. The data are representative of three experiments. ( b ) Exogenously expressed PTEN and NDRG2 were co-immunoprecipitated in 293T cells (WB, western blot). The data are representative of three experiments. ( c ) The co-immunoprecipitation of endogenous PTEN and NDRG2 was performed in MOLT4 cell lysates. On the input lane (−), 1/200 of the input was loaded for detection of NDRG2. Asterisk, nonspecific band. The data are representative of three experiments. ( d ) The co-localization of endogenous PTEN and NDRG2 was determined in MOLT4 cells. The nuclei were labelled with DAPI. Scale bar, 10 μm. The data are representative of three experiments.

    Article Snippet: Cell extracts from the KK1-NDRG2 stable cell line, NIH3T3 cell line and 293T cell line transiently transfected with mock or Myc-tagged PP1c, PP2Ac or PP5c vectors using HilyMax were prepared by lysing cells in TNT buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    Fibulin-5 interacts with LOXL enzymes. (A) Domain structures of the full-length fibulin-5 and the fibulin-5 deletion mutants used for in vitro binding assays. ΔN1-fibulin-5 corresponds to the naturally cleaved form of fibulin-5. These mutants were expressed as C-terminal-FLAG–tagged proteins. (B) Fibulin-5 binds to LOXL1, 2, and 4 through the C-terminal domain. 293T cells were transiently transfected with the vectors shown in A or a mock vector. Expression vectors for Myc-tagged LOX or LOXLs were also independently transfected into 293T cells. The conditioned media were harvested, and mixed. Each mixture was subjected to immunoprecipitation with anti-FLAG antibody, separated by SDS-PAGE, and analyzed by Western blotting with a monoclonal anti-Myc antibody.

    Journal: The Journal of Cell Biology

    Article Title: Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

    doi: 10.1083/jcb.200611026

    Figure Lengend Snippet: Fibulin-5 interacts with LOXL enzymes. (A) Domain structures of the full-length fibulin-5 and the fibulin-5 deletion mutants used for in vitro binding assays. ΔN1-fibulin-5 corresponds to the naturally cleaved form of fibulin-5. These mutants were expressed as C-terminal-FLAG–tagged proteins. (B) Fibulin-5 binds to LOXL1, 2, and 4 through the C-terminal domain. 293T cells were transiently transfected with the vectors shown in A or a mock vector. Expression vectors for Myc-tagged LOX or LOXLs were also independently transfected into 293T cells. The conditioned media were harvested, and mixed. Each mixture was subjected to immunoprecipitation with anti-FLAG antibody, separated by SDS-PAGE, and analyzed by Western blotting with a monoclonal anti-Myc antibody.

    Article Snippet: Cell culture 293T cells and human skin fibroblasts (HSFs) were maintained in DME (Sigma-Aldrich) supplemented with 2 mM glutamine, 10% penicillin/streptomycin, and 10% FBS at 37°C in 5% CO2 .

    Techniques: In Vitro, Binding Assay, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Western Blot

    Fibulin-5 is cleaved after the arginine at position 77 by serine protease and loses the microfibril-associating activity. (A) 293T cells were stably transfected with an expression vector encoding C-terminal-FLAG– and 6× histidine-tagged fibulin-5 cDNA. Recombinant fibulin-5 protein was purified by chelating chromatography from the culture media of these cells, subjected to SDS-PAGE, and stained with Coomassie blue. The lower band (arrow) was subjected to N-terminal sequencing. (B) The N-terminal sequencing of the lower band identified the specific cleavage site of fibulin-5 at the arginine at position 77. (C) 293T cells were transiently transfected with the expression vector encoding C-terminal-FLAG– and 6× histidine-tagged fibulin-5 or R77A mutant fibulin-5, which had a mutation from arginine to alanine at position 77. Transfected cells were cultured for 2 d with or without a cysteine protease inhibitor, E64, or a serine protease inhibitor, aprotinine, added to the culture media. Culture media were then harvested, concentrated by chelating chromatography, and subjected to Western blotting with anti-FLAG antibody. (D–G) Human skin fibroblasts were cultured for 4 d in serum-free medium in the presence of recombinant fibulin-5 (D) or recombinant cleaved fibulin-5 (E) proteins at a concentration of 4 μg/ml in the medium, or without recombinant protein (F). The cells were then double-stained with anti–fibrillin-1 polyclonal antibody (top) and with anti-FLAG monoclonal antibody (middle). Bottom images were produced by superimposition of the top and middle images, together with DAPI nuclear staining. Bars, 60 μm. At the same time, the conditioned medium was immunoblotted with anti-FLAG antibody to confirm that neither fibulin-5 nor cleaved fibulin-5 was degraded during the culture period (G).

    Journal: The Journal of Cell Biology

    Article Title: Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

    doi: 10.1083/jcb.200611026

    Figure Lengend Snippet: Fibulin-5 is cleaved after the arginine at position 77 by serine protease and loses the microfibril-associating activity. (A) 293T cells were stably transfected with an expression vector encoding C-terminal-FLAG– and 6× histidine-tagged fibulin-5 cDNA. Recombinant fibulin-5 protein was purified by chelating chromatography from the culture media of these cells, subjected to SDS-PAGE, and stained with Coomassie blue. The lower band (arrow) was subjected to N-terminal sequencing. (B) The N-terminal sequencing of the lower band identified the specific cleavage site of fibulin-5 at the arginine at position 77. (C) 293T cells were transiently transfected with the expression vector encoding C-terminal-FLAG– and 6× histidine-tagged fibulin-5 or R77A mutant fibulin-5, which had a mutation from arginine to alanine at position 77. Transfected cells were cultured for 2 d with or without a cysteine protease inhibitor, E64, or a serine protease inhibitor, aprotinine, added to the culture media. Culture media were then harvested, concentrated by chelating chromatography, and subjected to Western blotting with anti-FLAG antibody. (D–G) Human skin fibroblasts were cultured for 4 d in serum-free medium in the presence of recombinant fibulin-5 (D) or recombinant cleaved fibulin-5 (E) proteins at a concentration of 4 μg/ml in the medium, or without recombinant protein (F). The cells were then double-stained with anti–fibrillin-1 polyclonal antibody (top) and with anti-FLAG monoclonal antibody (middle). Bottom images were produced by superimposition of the top and middle images, together with DAPI nuclear staining. Bars, 60 μm. At the same time, the conditioned medium was immunoblotted with anti-FLAG antibody to confirm that neither fibulin-5 nor cleaved fibulin-5 was degraded during the culture period (G).

    Article Snippet: Cell culture 293T cells and human skin fibroblasts (HSFs) were maintained in DME (Sigma-Aldrich) supplemented with 2 mM glutamine, 10% penicillin/streptomycin, and 10% FBS at 37°C in 5% CO2 .

    Techniques: Activity Assay, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Recombinant, Purification, Chromatography, SDS Page, Staining, Sequencing, Mutagenesis, Cell Culture, Protease Inhibitor, Western Blot, Concentration Assay, Produced

    The N-terminal domain cleavage of fibulin-5 is found in aged mouse skin, as well as in cell cultures. (A) Fine structure of elastic fibers in skin tissues observed by transmission electromicroscopy. Elastin was stained with tannic acid, and therefore appears as black amorphous material. The fine fibers surrounding elastin are microfibrils. Bar, 0.4 μm. (B) Skin tissues were harvested from wild-type or fibulin-5–deficient young (3-mo-old) and old (22-mo-old) mice. Proteins were extracted from skin tissues with 8 M urea and dialyzed against PBS. 10 μg each of these extracts were resolved by SDS-PAGE, and analyzed by Western blotting with anti–fibulin-5 antibody (BSYN2473). Two specific bands of 45 and 55 kD were detected in wild-type mice with anti–fibulin-5 antibody (lanes 1–3). The 55-kD band markedly decreased with age, whereas the 45-kD band markedly increased with age (lanes 4–6). (C) 293T cells were transiently transfected with an expression vector encoding fibulin-5 cDNA with a signal peptide and a FLAG tag at the N terminus. Conditioned medium was subjected to SDS-PAGE, followed by Western blotting analysis with either anti– fibulin-5 or anti-FLAG antibody. Two bands of 55 and 45 kD were detected with anti–fibulin-5 antibody, whereas only a 55-kD band was detected with anti-FLAG antibody. (D) Anti–fibulin-5 antibody (BSYN2473) was raised against a peptide corresponding to amino acids 76–98 (red mark). These findings suggest that fibulin-5 is cleaved at a more N-terminal position than the recognition site of anti–fibulin-5 antibody.

    Journal: The Journal of Cell Biology

    Article Title: Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

    doi: 10.1083/jcb.200611026

    Figure Lengend Snippet: The N-terminal domain cleavage of fibulin-5 is found in aged mouse skin, as well as in cell cultures. (A) Fine structure of elastic fibers in skin tissues observed by transmission electromicroscopy. Elastin was stained with tannic acid, and therefore appears as black amorphous material. The fine fibers surrounding elastin are microfibrils. Bar, 0.4 μm. (B) Skin tissues were harvested from wild-type or fibulin-5–deficient young (3-mo-old) and old (22-mo-old) mice. Proteins were extracted from skin tissues with 8 M urea and dialyzed against PBS. 10 μg each of these extracts were resolved by SDS-PAGE, and analyzed by Western blotting with anti–fibulin-5 antibody (BSYN2473). Two specific bands of 45 and 55 kD were detected in wild-type mice with anti–fibulin-5 antibody (lanes 1–3). The 55-kD band markedly decreased with age, whereas the 45-kD band markedly increased with age (lanes 4–6). (C) 293T cells were transiently transfected with an expression vector encoding fibulin-5 cDNA with a signal peptide and a FLAG tag at the N terminus. Conditioned medium was subjected to SDS-PAGE, followed by Western blotting analysis with either anti– fibulin-5 or anti-FLAG antibody. Two bands of 55 and 45 kD were detected with anti–fibulin-5 antibody, whereas only a 55-kD band was detected with anti-FLAG antibody. (D) Anti–fibulin-5 antibody (BSYN2473) was raised against a peptide corresponding to amino acids 76–98 (red mark). These findings suggest that fibulin-5 is cleaved at a more N-terminal position than the recognition site of anti–fibulin-5 antibody.

    Article Snippet: Cell culture 293T cells and human skin fibroblasts (HSFs) were maintained in DME (Sigma-Aldrich) supplemented with 2 mM glutamine, 10% penicillin/streptomycin, and 10% FBS at 37°C in 5% CO2 .

    Techniques: Transmission Assay, Staining, Mouse Assay, SDS Page, Western Blot, Transfection, Expressing, Plasmid Preparation, FLAG-tag

    TIN2 is phosphorylated by the mitotic kinase RSK2. ( A ) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. ( B ) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. ( C ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. ( D ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2 Y707A ) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left ( top ). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control ( bottom ). Representative of two experiments. ( E ) Detection of TIN2 phosphorylation by RSK2 in vitro . Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP 32 , after which the reaction products were resolved by SDS-PAGE and either ( top ) exposed to autographic film or ( bottom ) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P 32 ) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.

    Journal: PLoS ONE

    Article Title: Cell Cycle Regulated Phosphorylation of the Telomere-Associated Protein TIN2

    doi: 10.1371/journal.pone.0071697

    Figure Lengend Snippet: TIN2 is phosphorylated by the mitotic kinase RSK2. ( A ) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. ( B ) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. ( C ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. ( D ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2 Y707A ) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left ( top ). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control ( bottom ). Representative of two experiments. ( E ) Detection of TIN2 phosphorylation by RSK2 in vitro . Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP 32 , after which the reaction products were resolved by SDS-PAGE and either ( top ) exposed to autographic film or ( bottom ) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P 32 ) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.

    Article Snippet: In some cases, HeLa or 293T cell lines were treated with 10 µM H-89 (Sigma), 10 µM BI-D1870 (Enzo Life Science), 10 nM BI 2536 (Selleckbio), or 1 µM VX-680 (Selleckbio) for 30 minutes in the presence of nocodazole before harvesting.

    Techniques: Stable Transfection, Expressing, Derivative Assay, Immunoprecipitation, SDS Page, Staining, Fluorescence, FACS, Transfection, Mutagenesis, Activity Assay, In Vitro, Recombinant, Binding Assay, Purification, Incubation

    The involvement of Vps4A and Vps4B in HTLV-1 Gag budding . A. 293T cells were cotransfected with pK30-Gag and the expression plasmid for Vps4AEQ or Vps4BEQ, or the empty vector as a control. Extracellular VLPs were pelleted from the culture fluids. VLP-associated or cell-associated Gag was detected by western blotting (WB) using anti-HTLV-1 p19 monoclonal antibody. C. 293T cells were cotransfected with pK30-Gag and the expression vector for wild-type Vps4A or Vps4B, or the empty vector as a control. The proteins were detected as described in A. B and D. Intensities of the bands corresponding to cell- and VLP-associated Gag in A and C were quantified using the LAS3000 imaging system (Fuji film). The efficiency of Gag-induced VLP budding in cells cotransfected with pK30-Gag and control vector (VLP/Cellular) was set to 1.0. The data represent averages and standard deviations (SD) of 3 independent experiments.

    Journal: Virology Journal

    Article Title: Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

    doi: 10.1186/1743-422X-4-66

    Figure Lengend Snippet: The involvement of Vps4A and Vps4B in HTLV-1 Gag budding . A. 293T cells were cotransfected with pK30-Gag and the expression plasmid for Vps4AEQ or Vps4BEQ, or the empty vector as a control. Extracellular VLPs were pelleted from the culture fluids. VLP-associated or cell-associated Gag was detected by western blotting (WB) using anti-HTLV-1 p19 monoclonal antibody. C. 293T cells were cotransfected with pK30-Gag and the expression vector for wild-type Vps4A or Vps4B, or the empty vector as a control. The proteins were detected as described in A. B and D. Intensities of the bands corresponding to cell- and VLP-associated Gag in A and C were quantified using the LAS3000 imaging system (Fuji film). The efficiency of Gag-induced VLP budding in cells cotransfected with pK30-Gag and control vector (VLP/Cellular) was set to 1.0. The data represent averages and standard deviations (SD) of 3 independent experiments.

    Article Snippet: Cells Human 293T cells were maintained in Dulbecco's minimal essential medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37°C.

    Techniques: Expressing, Plasmid Preparation, Western Blot, Imaging

    Effects of AIP1/Alix DN mutants on HTLV-1 VLP production . A. 293T cells were cotransfected with pK30-Gag and the expression plasmid for AIP1 (1–628), AIP1 (424–628), or AIP1 WT, or the empty vector as a control. Extracellular VLPs were pelleted from the culture fluids. VLP- or cell-associated Gag was detected by WB using anti-p19 monoclonal antibody. B. Intensities of the bands corresponding to cell- and VLP-associated Gag in A were quantified using the LAS3000 imaging system (Fuji Film). The efficiency of Gag-induced VLP budding in cells cotransfected with pK30-Gag and the control vector (VLP/Cellular) was set to 1.0. The data represent averages and standard deviations (SD) of 3 independent experiments.

    Journal: Virology Journal

    Article Title: Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

    doi: 10.1186/1743-422X-4-66

    Figure Lengend Snippet: Effects of AIP1/Alix DN mutants on HTLV-1 VLP production . A. 293T cells were cotransfected with pK30-Gag and the expression plasmid for AIP1 (1–628), AIP1 (424–628), or AIP1 WT, or the empty vector as a control. Extracellular VLPs were pelleted from the culture fluids. VLP- or cell-associated Gag was detected by WB using anti-p19 monoclonal antibody. B. Intensities of the bands corresponding to cell- and VLP-associated Gag in A were quantified using the LAS3000 imaging system (Fuji Film). The efficiency of Gag-induced VLP budding in cells cotransfected with pK30-Gag and the control vector (VLP/Cellular) was set to 1.0. The data represent averages and standard deviations (SD) of 3 independent experiments.

    Article Snippet: Cells Human 293T cells were maintained in Dulbecco's minimal essential medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37°C.

    Techniques: Expressing, Plasmid Preparation, Western Blot, Imaging

    Splicing assays of wild-type and mutant PRPF31 in transfected HEK 293T cells. A: Assays using RHO intron 3 minigene splicing template ( RHO -MG). As a positive control, cells were transfected with the splicing template only ( RHO -MG only). Cells transfected with WT PRPF31 only gave no products (negative control). Marker for the unspliced product was generated by amplification directly from the plasmid construct using the same primers. B: Bar graphs show the splicing efficiencies, derived from the relative band strengths for cells expressing untagged and His-tagged PRPF31. Error bars indicate the standard error of means derived from four separate determinations. Double asterisks (**) indicate that the reduced splicing efficiencies of mutant splicing factor compared to the positive and negative controls are statistically significant (p

    Journal: Molecular Vision

    Article Title: Disease mechanism for retinitis pigmentosa (RP11) caused by missense mutations in the splicing factor gene PRPF31

    doi:

    Figure Lengend Snippet: Splicing assays of wild-type and mutant PRPF31 in transfected HEK 293T cells. A: Assays using RHO intron 3 minigene splicing template ( RHO -MG). As a positive control, cells were transfected with the splicing template only ( RHO -MG only). Cells transfected with WT PRPF31 only gave no products (negative control). Marker for the unspliced product was generated by amplification directly from the plasmid construct using the same primers. B: Bar graphs show the splicing efficiencies, derived from the relative band strengths for cells expressing untagged and His-tagged PRPF31. Error bars indicate the standard error of means derived from four separate determinations. Double asterisks (**) indicate that the reduced splicing efficiencies of mutant splicing factor compared to the positive and negative controls are statistically significant (p

    Article Snippet: In vivo splicing assays in HEK 293T cells Human HEK 293T cells in 3.5 cm dishes were transiently transfected using GeneJuice (Novagen) with pTandem-1 constructs which expressed PRPF31 (WT or mutant) and a splicing template comprising either the RHO intron 3 mini-gene, the RHO full length gene, or the GNAT1 partial gene.

    Techniques: Mutagenesis, Transfection, Positive Control, Negative Control, Marker, Generated, Amplification, Plasmid Preparation, Construct, Derivative Assay, Expressing

    p27 is SUMOylated on K134 in vitro and in vivo . ( A ) Western blot analysis of FLAG-p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or Flag-p27 1–170 , in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. Asterisk indicates the IgG heavy chain band. ( B ) Western blot analysis of p27 WT recombinant protein in an in vitro SUMOylation assay. His-tagged p27 was incubated with SAE1/SAE2, SUMO, and increasing doses of Ubc9. p27 SUMOylation, expressed as a SUMOylated p27/p27 ratio, was calculated by densitometric analysis of the blots. ( C ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence or absence of HA-SUMO1 and untagged Ubc9. ( D ) Western blot analysis of p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. ( E ) Western blot analysis of p27 and SUMO1 expression in SUMO1 (IP SUMO1) or control (IP IgG) immunoprecipitates from HeLa cells transfected with FLAG-p27 WT or FLAG-p27 K134R in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate SUMO-modified p27 proteins detected using anti-p27 antibody (upper panel) or the immunoprecipitated SUMO1 (lower panel). Asterisk indicates the IgG heavy chain band. ( F ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) and immunoprecipitates (IP) derived from 293T/17 cells transfected with untagged p27 WT in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-p27 (lower and middle panels) or anti-HA (upper panel) antibodies.

    Journal: Journal of Molecular Cell Biology

    Article Title: SUMOylation regulates p27Kip1 stability and localization in response to TGFβ

    doi: 10.1093/jmcb/mjv056

    Figure Lengend Snippet: p27 is SUMOylated on K134 in vitro and in vivo . ( A ) Western blot analysis of FLAG-p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or Flag-p27 1–170 , in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. Asterisk indicates the IgG heavy chain band. ( B ) Western blot analysis of p27 WT recombinant protein in an in vitro SUMOylation assay. His-tagged p27 was incubated with SAE1/SAE2, SUMO, and increasing doses of Ubc9. p27 SUMOylation, expressed as a SUMOylated p27/p27 ratio, was calculated by densitometric analysis of the blots. ( C ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence or absence of HA-SUMO1 and untagged Ubc9. ( D ) Western blot analysis of p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. ( E ) Western blot analysis of p27 and SUMO1 expression in SUMO1 (IP SUMO1) or control (IP IgG) immunoprecipitates from HeLa cells transfected with FLAG-p27 WT or FLAG-p27 K134R in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate SUMO-modified p27 proteins detected using anti-p27 antibody (upper panel) or the immunoprecipitated SUMO1 (lower panel). Asterisk indicates the IgG heavy chain band. ( F ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) and immunoprecipitates (IP) derived from 293T/17 cells transfected with untagged p27 WT in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-p27 (lower and middle panels) or anti-HA (upper panel) antibodies.

    Article Snippet: HeLa (human cervical adenocarcinoma cell line, ATCC CCL-2), MCF7 (human breast adenocarcinoma cell line, ATCC HTB-22), and 293T/17 (human embryonic kidney cell line) cells were grown in DMEM supplemented with 10% heat-inactivated FBS (Sigma).

    Techniques: In Vitro, In Vivo, Western Blot, Expressing, Derivative Assay, Transfection, Modification, Recombinant, Incubation, Immunoprecipitation

    TGFβ-induced SUMOylation decreases p27 affinity for CDK2. ( A ) In vivo ubiquitination assay in 293T/17 cells transfected with the indicated FLAG-tagged vectors and with HA-tagged ubiquitin. Cells were then treated or not with TGFβ (20 ng/ml). Lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-HA and anti-p27 antibodies. Cell lysates were probed with anti-HA antibody for the equivalent level of ubiquitin transfection in different samples (lower panel). ( B ) In vivo ubiquitination assay in 293T/17 cells transfected with the indicated FLAG-tagged vectors along with HA-tagged ubiquitin. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) antibody and probed with anti-HA and anti-p27 antibodies. Cell lysates were probed with anti-HA antibody for the equivalent level of ubiquitin transfection in different samples (lower panel). ( C ) Co-immunoprecipitation analysis (IP: FLAG) of protein lysates derived from MCF7 cells transfected with FLAG-p27 WT , FLAG-p27 K134R , or FLAG empty vector in the presence of HA-SUMO1 and untagged Ubc9, and treated or not with TGFβ (20 ng/ml) for 3 h. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) resin and probed with anti-FLAG and anti-CDK2 antibodies. Lysates (Input) were analyzed for the expression of CDK2, FLAG-p27, and Smad2 phosphorylated on Ser 465/467 (pSmad2) to confirm the activation of the TGFβ pathway. Vinculin was used as loading control. The FLAG-p27/CDK2 interaction is reported as the ratio of FLAG-p27 WT bound to CDK2 based on densitometric analysis of the blots. ( D ) Co-immunoprecipitation analysis (IP: FLAG) of protein lysates derived from HeLa cells transfected with FLAG-p27 WT , FLAG-p27 K134R , or FLAG empty vector in the presence of HA-SUMO1 and untagged Ubc9, and treated or not with TGFβ (20 ng/ml) for 3 h. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) resin and probed with anti-FLAG, anti-Skp2, and anti-CDK2 antibodies.

    Journal: Journal of Molecular Cell Biology

    Article Title: SUMOylation regulates p27Kip1 stability and localization in response to TGFβ

    doi: 10.1093/jmcb/mjv056

    Figure Lengend Snippet: TGFβ-induced SUMOylation decreases p27 affinity for CDK2. ( A ) In vivo ubiquitination assay in 293T/17 cells transfected with the indicated FLAG-tagged vectors and with HA-tagged ubiquitin. Cells were then treated or not with TGFβ (20 ng/ml). Lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-HA and anti-p27 antibodies. Cell lysates were probed with anti-HA antibody for the equivalent level of ubiquitin transfection in different samples (lower panel). ( B ) In vivo ubiquitination assay in 293T/17 cells transfected with the indicated FLAG-tagged vectors along with HA-tagged ubiquitin. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) antibody and probed with anti-HA and anti-p27 antibodies. Cell lysates were probed with anti-HA antibody for the equivalent level of ubiquitin transfection in different samples (lower panel). ( C ) Co-immunoprecipitation analysis (IP: FLAG) of protein lysates derived from MCF7 cells transfected with FLAG-p27 WT , FLAG-p27 K134R , or FLAG empty vector in the presence of HA-SUMO1 and untagged Ubc9, and treated or not with TGFβ (20 ng/ml) for 3 h. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) resin and probed with anti-FLAG and anti-CDK2 antibodies. Lysates (Input) were analyzed for the expression of CDK2, FLAG-p27, and Smad2 phosphorylated on Ser 465/467 (pSmad2) to confirm the activation of the TGFβ pathway. Vinculin was used as loading control. The FLAG-p27/CDK2 interaction is reported as the ratio of FLAG-p27 WT bound to CDK2 based on densitometric analysis of the blots. ( D ) Co-immunoprecipitation analysis (IP: FLAG) of protein lysates derived from HeLa cells transfected with FLAG-p27 WT , FLAG-p27 K134R , or FLAG empty vector in the presence of HA-SUMO1 and untagged Ubc9, and treated or not with TGFβ (20 ng/ml) for 3 h. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) resin and probed with anti-FLAG, anti-Skp2, and anti-CDK2 antibodies.

    Article Snippet: HeLa (human cervical adenocarcinoma cell line, ATCC CCL-2), MCF7 (human breast adenocarcinoma cell line, ATCC HTB-22), and 293T/17 (human embryonic kidney cell line) cells were grown in DMEM supplemented with 10% heat-inactivated FBS (Sigma).

    Techniques: In Vivo, Ubiquitin Assay, Transfection, Immunoprecipitation, Derivative Assay, Plasmid Preparation, Expressing, Activation Assay

    APRIN silencing increases genomic instability and heightens sensitivity to DNA-damaging agents. ( A – C ) Survival curves of 293T cells transfected with siRNA targeting APRIN and then treated with either ( A ) aphidicholin or ( B ) HU or ( C ) MMC for 5

    Journal: The EMBO Journal

    Article Title: APRIN is a cell cycle specific BRCA2-interacting protein required for genome integrity and a predictor of outcome after chemotherapy in breast cancer

    doi: 10.1038/emboj.2011.490

    Figure Lengend Snippet: APRIN silencing increases genomic instability and heightens sensitivity to DNA-damaging agents. ( A – C ) Survival curves of 293T cells transfected with siRNA targeting APRIN and then treated with either ( A ) aphidicholin or ( B ) HU or ( C ) MMC for 5

    Article Snippet: Human 293T, Hela and T47D cells were cultured and maintained at 37°C (5% CO2 ) in Dulbecco's modified Eagle's medium (Sigma, Poole, UK) supplemented with FBS (PAA), 1% L -glutamine and penicillin–streptomycin (Gibco).

    Techniques: Transfection

    The BRCA2/APRIN interaction is cell-cycle dependent. ( A ) Western blot analysis of BRCA2 immunoprecipitated material or whole cell lysates (WCL) from AS or synchronous 293T cultures. For synchronous cultures, cells were arrested at the G 1 /S checkpoint

    Journal: The EMBO Journal

    Article Title: APRIN is a cell cycle specific BRCA2-interacting protein required for genome integrity and a predictor of outcome after chemotherapy in breast cancer

    doi: 10.1038/emboj.2011.490

    Figure Lengend Snippet: The BRCA2/APRIN interaction is cell-cycle dependent. ( A ) Western blot analysis of BRCA2 immunoprecipitated material or whole cell lysates (WCL) from AS or synchronous 293T cultures. For synchronous cultures, cells were arrested at the G 1 /S checkpoint

    Article Snippet: Human 293T, Hela and T47D cells were cultured and maintained at 37°C (5% CO2 ) in Dulbecco's modified Eagle's medium (Sigma, Poole, UK) supplemented with FBS (PAA), 1% L -glutamine and penicillin–streptomycin (Gibco).

    Techniques: Western Blot, Immunoprecipitation

    APRIN is involved in DNA repair. ( A ) (Left panel) Representative confocal microscopy images from 293T cells transfected with non-targeting control siRNA or siRNA targeting APRIN. Forty-eight hours after transfection, cells were exposed to 10 Gy IR and

    Journal: The EMBO Journal

    Article Title: APRIN is a cell cycle specific BRCA2-interacting protein required for genome integrity and a predictor of outcome after chemotherapy in breast cancer

    doi: 10.1038/emboj.2011.490

    Figure Lengend Snippet: APRIN is involved in DNA repair. ( A ) (Left panel) Representative confocal microscopy images from 293T cells transfected with non-targeting control siRNA or siRNA targeting APRIN. Forty-eight hours after transfection, cells were exposed to 10 Gy IR and

    Article Snippet: Human 293T, Hela and T47D cells were cultured and maintained at 37°C (5% CO2 ) in Dulbecco's modified Eagle's medium (Sigma, Poole, UK) supplemented with FBS (PAA), 1% L -glutamine and penicillin–streptomycin (Gibco).

    Techniques: Confocal Microscopy, Transfection

    BRCA2 and APRIN associate with cohesion proteins and replication complex. ( A ) Western blot analysis of BRCA2 immunoprecipitated material or whole cell lysates (WCL) from AS or synchronous 293T cultures. For synchronous cultures, cells were arrested at

    Journal: The EMBO Journal

    Article Title: APRIN is a cell cycle specific BRCA2-interacting protein required for genome integrity and a predictor of outcome after chemotherapy in breast cancer

    doi: 10.1038/emboj.2011.490

    Figure Lengend Snippet: BRCA2 and APRIN associate with cohesion proteins and replication complex. ( A ) Western blot analysis of BRCA2 immunoprecipitated material or whole cell lysates (WCL) from AS or synchronous 293T cultures. For synchronous cultures, cells were arrested at

    Article Snippet: Human 293T, Hela and T47D cells were cultured and maintained at 37°C (5% CO2 ) in Dulbecco's modified Eagle's medium (Sigma, Poole, UK) supplemented with FBS (PAA), 1% L -glutamine and penicillin–streptomycin (Gibco).

    Techniques: Western Blot, Immunoprecipitation

    Direct binding measurements of CD4-Ig and mabs follows neutralization sensitivity of Env+ pseudoviruses. LN8 wt, 375W and 380P Envs were expressed on 293T cells before measuring binding of CD4-Ig and mabs using flow cytometry. Boxed values in the right hand, top corner of each flow profile represents the neutralization titer for each reagent and shows that binding closely followed neutralization sensitivity.

    Journal: PLoS Pathogens

    Article Title: Saturation Mutagenesis of the HIV-1 Envelope CD4 Binding Loop Reveals Residues Controlling Distinct Trimer Conformations

    doi: 10.1371/journal.ppat.1005988

    Figure Lengend Snippet: Direct binding measurements of CD4-Ig and mabs follows neutralization sensitivity of Env+ pseudoviruses. LN8 wt, 375W and 380P Envs were expressed on 293T cells before measuring binding of CD4-Ig and mabs using flow cytometry. Boxed values in the right hand, top corner of each flow profile represents the neutralization titer for each reagent and shows that binding closely followed neutralization sensitivity.

    Article Snippet: Expression of Env trimers on 293T cells and mab binding HIV-1 Envs were expressed on 293T cells following transfection of Env expression vectors using Fugene6 following the manufacturer’s protocol.

    Techniques: Binding Assay, Neutralization, Flow Cytometry, Cytometry

    Chimeric antigen receptors (CAR) constructs for CD8+ T cells transduction ( A ) T cells were transduced with the lentiviruses to generate anti-CAIX CAR T cells, which are able to recognize CAIX positive RCC, and also secrete anti-PD-L1 IgG1 or IgG4 in the tumor microenvironment to block PD-1/PD-L1-induced T cell exhaustion. ( B ) Schematic representation of pHAGE lentiviral vectors encoding second-generation CARs fused with CD28 co-stimulatory endodomain. The anti-carbonic anhydrase IX (CAIX) or the Anti-B-cell maturation antigen (BCMA) scFv (as a negative control) were inserted after the eIFa promoter in order to express the CAR binding domain. The second cassette, after the Internal Ribosome Entry Site (IRES) sequence, encodes the secretable anti-PD-L1 IgG1 or IgG4 isotypes or the anti-severe acute respiratory syndrome (SARS) coronavirus IgG1 (negative control). LTR: long terminal repeat, eIFα: eukaryotic initiation factor alpha, scFv: single-chain variable fragment, C9 TAG: C9 peptide TETSQVAPA, IRES: Internal Ribosome Entry Site, WPRE: Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. ( C ) Percentage of CAR T cells 14 days after transduction, representing the stable long-term expression of CAR by the integrated lentiviruses in CD8+ T cells. The CD8+ T cells were selected using Dynabeads CD8 Positive Isolation Kit (Life Technologies) and activated with Dynabeads Human T Cell Activator CD3/CD28 (Life Technologies) in the presence of IL-21 50 U/mL. IL-21 was added to the medium every 2 days. After 14 days, the CAR T cells were incubated with human CAIX-Fc or BCMA-Fc, followed by incubation with an APC conjugated anti-human Fc IgG (Southern Biotech) or goat-anti-mouse IgG Ab (Biolegend) and analyzed by flow cytometry. ( D ) Concentration of IgG secreted into the medium of transduced T cells evaluated by Human IgG ELISA Quantitation Set (Bethyl Laboratories). ( E ) Concentration of anti-PD-L1 antibodies in the supernatant of 293T Cells transduced with lentiviruses containing anti-CAIX or anti-BCMA CAR and anti-PD-L1 IgG1, anti-PD-L1 IgG4 or irrelevant anti-SARS IgG1 sequences. The antibodies in the supernatant were purified, biotinylated and incubated with 5 μg/mL of human PD-L1 pre-immobilized in the 96 wells MaxiSorp plate (Nunc). The biotinylated antibodies were detected by incubation with streptavidin-HRP and developed with TMB. The absorbance was read at λ = 450 nm. * P

    Journal: Oncotarget

    Article Title: Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model

    doi: 10.18632/oncotarget.9114

    Figure Lengend Snippet: Chimeric antigen receptors (CAR) constructs for CD8+ T cells transduction ( A ) T cells were transduced with the lentiviruses to generate anti-CAIX CAR T cells, which are able to recognize CAIX positive RCC, and also secrete anti-PD-L1 IgG1 or IgG4 in the tumor microenvironment to block PD-1/PD-L1-induced T cell exhaustion. ( B ) Schematic representation of pHAGE lentiviral vectors encoding second-generation CARs fused with CD28 co-stimulatory endodomain. The anti-carbonic anhydrase IX (CAIX) or the Anti-B-cell maturation antigen (BCMA) scFv (as a negative control) were inserted after the eIFa promoter in order to express the CAR binding domain. The second cassette, after the Internal Ribosome Entry Site (IRES) sequence, encodes the secretable anti-PD-L1 IgG1 or IgG4 isotypes or the anti-severe acute respiratory syndrome (SARS) coronavirus IgG1 (negative control). LTR: long terminal repeat, eIFα: eukaryotic initiation factor alpha, scFv: single-chain variable fragment, C9 TAG: C9 peptide TETSQVAPA, IRES: Internal Ribosome Entry Site, WPRE: Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. ( C ) Percentage of CAR T cells 14 days after transduction, representing the stable long-term expression of CAR by the integrated lentiviruses in CD8+ T cells. The CD8+ T cells were selected using Dynabeads CD8 Positive Isolation Kit (Life Technologies) and activated with Dynabeads Human T Cell Activator CD3/CD28 (Life Technologies) in the presence of IL-21 50 U/mL. IL-21 was added to the medium every 2 days. After 14 days, the CAR T cells were incubated with human CAIX-Fc or BCMA-Fc, followed by incubation with an APC conjugated anti-human Fc IgG (Southern Biotech) or goat-anti-mouse IgG Ab (Biolegend) and analyzed by flow cytometry. ( D ) Concentration of IgG secreted into the medium of transduced T cells evaluated by Human IgG ELISA Quantitation Set (Bethyl Laboratories). ( E ) Concentration of anti-PD-L1 antibodies in the supernatant of 293T Cells transduced with lentiviruses containing anti-CAIX or anti-BCMA CAR and anti-PD-L1 IgG1, anti-PD-L1 IgG4 or irrelevant anti-SARS IgG1 sequences. The antibodies in the supernatant were purified, biotinylated and incubated with 5 μg/mL of human PD-L1 pre-immobilized in the 96 wells MaxiSorp plate (Nunc). The biotinylated antibodies were detected by incubation with streptavidin-HRP and developed with TMB. The absorbance was read at λ = 450 nm. * P

    Article Snippet: Briefly, each 80% confluent 293T cells in 15 cm plate (Nalge Nunc) was transfected with 30 μg of total five plasmids, being 5 μg of each structural plasmid pHDH-Hgpm2 (HIV gag-pol), pMD-tat; pRC/CMV-rev and Env VSV-G, and 10 μg of the CAR encoding plasmid (anti-CAIX/anti-PD-L1 IgG1, anti-CAIX/anti-PD-L1 IgG4, anti-CAIX/anti-SARS IgG1 or anti-BCMA/anti-SARS IgG1).

    Techniques: Construct, Transduction, Blocking Assay, Negative Control, Binding Assay, Sequencing, Expressing, Isolation, Incubation, Flow Cytometry, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Purification

    Socs-1 inhibits kinase activity of TEL-JAK2. (A) TEL-JAK2 in vitro kinase assay. 293T cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc–Socs-1 were starved of FBS for 4 h. Extracts from these cells were immunoprecipitated (IP) with a TEL antibody and used for in vitro kinase assays using GST-C′Gab2 as a substrate. Products were separated by SDS-PAGE and visualized by autoradiography (top). Parallel TEL Western blotting confirmed equal immunoprecipitation of the protein (bottom). (B) 4G10 Western blot. 293T cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc-Socs-1 were starved of FBS for 4 h. TEL-JAK2 was immunoprecipitated with the JAK2 antibody and blotted with 4G10 (top) or JAK2 antibody (bottom). The whole-cell lysates were blotted with a Myc antibody to confirm expression of Socs-1 and a TEL antibody to confirm equal expression of TEL-JAK2 (data not shown). (C) Phosphoamino acid analysis of C′Gab2. Phosphorylated GST-C′Gab2 substrate from panel was hydrolyzed to single amino acids and separated on a thin-layer chromatography plate (left). The plate was placed on a phosphorimager, and the ratio of phosphorylated amino acids was used to generate the graph (right). Open bars represent TEL-JAK2 samples, and solid bars represent Socs-1 and TEL-JAK2 samples. (D) Phosphoamino acid analysis of autophosphorylated TEL-JAK2. Analysis of the autophosphorylated TEL-JAK2 was performed as for panel C. Open bars represent TEL-JAK2 samples, and solid bars represent Socs-1 and TEL-JAK2 samples.

    Journal: Molecular and Cellular Biology

    Article Title: Socs-1 Inhibits TEL-JAK2-Mediated Transformation of Hematopoietic Cells through Inhibition of JAK2 Kinase Activity and Induction of Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.10.3547-3557.2001

    Figure Lengend Snippet: Socs-1 inhibits kinase activity of TEL-JAK2. (A) TEL-JAK2 in vitro kinase assay. 293T cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc–Socs-1 were starved of FBS for 4 h. Extracts from these cells were immunoprecipitated (IP) with a TEL antibody and used for in vitro kinase assays using GST-C′Gab2 as a substrate. Products were separated by SDS-PAGE and visualized by autoradiography (top). Parallel TEL Western blotting confirmed equal immunoprecipitation of the protein (bottom). (B) 4G10 Western blot. 293T cells expressing 5/19 TEL-JAK2 and increasing amounts of Myc-Socs-1 were starved of FBS for 4 h. TEL-JAK2 was immunoprecipitated with the JAK2 antibody and blotted with 4G10 (top) or JAK2 antibody (bottom). The whole-cell lysates were blotted with a Myc antibody to confirm expression of Socs-1 and a TEL antibody to confirm equal expression of TEL-JAK2 (data not shown). (C) Phosphoamino acid analysis of C′Gab2. Phosphorylated GST-C′Gab2 substrate from panel was hydrolyzed to single amino acids and separated on a thin-layer chromatography plate (left). The plate was placed on a phosphorimager, and the ratio of phosphorylated amino acids was used to generate the graph (right). Open bars represent TEL-JAK2 samples, and solid bars represent Socs-1 and TEL-JAK2 samples. (D) Phosphoamino acid analysis of autophosphorylated TEL-JAK2. Analysis of the autophosphorylated TEL-JAK2 was performed as for panel C. Open bars represent TEL-JAK2 samples, and solid bars represent Socs-1 and TEL-JAK2 samples.

    Article Snippet: Retroviral stocks were generated by transient cotransfection of 293T cells with the appropriate MSCV constructs together with a packaging construct (pIK6.1MCV.ecopac.UTd; Cell Genesys Inc., Foster City, Calif.) providing sequences necessary for retrovirus production ( ).

    Techniques: Activity Assay, In Vitro, Kinase Assay, Expressing, Immunoprecipitation, SDS Page, Autoradiography, Western Blot, Phosphoamino Acid Analysis, Thin Layer Chromatography

    Socs-1 associates with TEL-JAK2 and inhibits tyrosine phosphorylation of STAT5. (A) Socs-1 associates with TEL-JAK2. Extracts from 293T cells expressing the 5/19 variant of TEL-JAK2 (1 μg) in the absence or presence of Socs-1 (1 μg) were immunoprecipitated (IP) with a JAK2 antibody and blotted with an anti-Myc antibody to detect the Myc-tagged Socs-1 (top). The membrane was blotted with a JAK2 antibody to confirm precipitation of the TEL-JAK2 (bottom). (B) Socs-1 expression results in decreased overall tyrosine phosphorylation of 293T cells expressing TEL-JAK2. Whole-cell extracts from cells transfected with 2.5 μg of 5/19 variant of TEL-JAK2 in the absence or presence of 10 μg of Socs-1 were blotted with 4G10 (top) or a Stat5 antibody to confirm equal loading of the lysates (bottom). The arrow identifies TEL-JAK2, which is seen as a doublet due to an alternative start site for translation in TEL. The whole-cell extracts were blotted with a Myc antibody to confirm expression of Socs-1 and a TEL antibody to confirm expression of TEL-JAK2 (data not shown). (C) Socs-1 inhibits tyrosine phosphorylation of Stat5 in 293T cells expressing TEL-JAK2. Extracts from 293T cells expressing the 5/19 TEL-JAK2 variant and increasing amounts Socs-1 were immunoprecipitated with a Stat5 antibody and blotted with 4G10 (top) or a Stat5 antibody (middle). Whole-cell lysates (WCL) were blotted with an anti-TEL antibody to confirm equal expression of TEL-JAK2 (bottom).

    Journal: Molecular and Cellular Biology

    Article Title: Socs-1 Inhibits TEL-JAK2-Mediated Transformation of Hematopoietic Cells through Inhibition of JAK2 Kinase Activity and Induction of Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.10.3547-3557.2001

    Figure Lengend Snippet: Socs-1 associates with TEL-JAK2 and inhibits tyrosine phosphorylation of STAT5. (A) Socs-1 associates with TEL-JAK2. Extracts from 293T cells expressing the 5/19 variant of TEL-JAK2 (1 μg) in the absence or presence of Socs-1 (1 μg) were immunoprecipitated (IP) with a JAK2 antibody and blotted with an anti-Myc antibody to detect the Myc-tagged Socs-1 (top). The membrane was blotted with a JAK2 antibody to confirm precipitation of the TEL-JAK2 (bottom). (B) Socs-1 expression results in decreased overall tyrosine phosphorylation of 293T cells expressing TEL-JAK2. Whole-cell extracts from cells transfected with 2.5 μg of 5/19 variant of TEL-JAK2 in the absence or presence of 10 μg of Socs-1 were blotted with 4G10 (top) or a Stat5 antibody to confirm equal loading of the lysates (bottom). The arrow identifies TEL-JAK2, which is seen as a doublet due to an alternative start site for translation in TEL. The whole-cell extracts were blotted with a Myc antibody to confirm expression of Socs-1 and a TEL antibody to confirm expression of TEL-JAK2 (data not shown). (C) Socs-1 inhibits tyrosine phosphorylation of Stat5 in 293T cells expressing TEL-JAK2. Extracts from 293T cells expressing the 5/19 TEL-JAK2 variant and increasing amounts Socs-1 were immunoprecipitated with a Stat5 antibody and blotted with 4G10 (top) or a Stat5 antibody (middle). Whole-cell lysates (WCL) were blotted with an anti-TEL antibody to confirm equal expression of TEL-JAK2 (bottom).

    Article Snippet: Retroviral stocks were generated by transient cotransfection of 293T cells with the appropriate MSCV constructs together with a packaging construct (pIK6.1MCV.ecopac.UTd; Cell Genesys Inc., Foster City, Calif.) providing sequences necessary for retrovirus production ( ).

    Techniques: Expressing, Variant Assay, Immunoprecipitation, Transfection

    TEL-JAK2 associates with Shc and Grb2 and activates Erk2. (A) Shc is tyrosine phosphorylated in Ba/F3 cells expressing the TEL-JAK2 fusion variants and associates with TEL-JAK2. Extracts from Ba/F3 cells expressing the TEL-JAK2 fusion variants were immunoprecipitated (IP) with an anti-Shc antibody and blotted with 4G10 (top). TEL-JAK2 is seen as a doublet due to an alternative start site for translation in TEL. The membrane was blotted with a Shc antibody to confirm equal precipitation of the protein (bottom). Control experiments showed no evidence of association of Shc with inactive TEL-JAK2 mutants that lacked the PNT domain or were kinase inactive (data not shown). (B) Grb2 associates with the 5/19 and 5/12 TEL-JAK2 fusion variants. Extracts from panel A were immunoprecipitated with a Grb2 antibody and blotted with a TEL antibody. Association of Grb2 with the 4/17 variant was not detected. Control experiments showed no evidence of association of Grb2 with inactive TEL-JAK2 mutants that lacked the PNT domain or were kinase inactive (data not shown). The membrane was blotted with a Grb2 antibody to confirm equal precipitation (data not shown). (C) The SH2 domain of Grb2 directly associates with the 5/19 and 5/12 TEL-JAK2 fusion variants. Extracts from 293T cells expressing empty vector or the 5/19 TEL-JAK2 fusion variant were immunoprecipitated with a TEL antibody. Far-Western analysis was performed with purified GST and blotted with a GST antibody (left). Extracts from 293T cells expressing the 5/19 and 5/12 TEL-JAK2 fusion variants were immunoprecipitated with a TEL antibody, and far-Western analysis was performed with GST–Grb2 SH2 (middle). The membrane was blotted with TEL antibody to confirm precipitation of 5/19 and 5/12 TEL-JAK2 (right). (D) The SH2 domain of Grb2 does not directly associate with the 4/17 fusion variant. Extracts from 293T cells expressing the 4/17 TEL-JAK2 fusion variant was immunoprecipitated with a TEL antibody. Far-Western analysis was performed with purified GST (data not shown) or GST-Grb2 SH2 and blotted with a GST antibody (left). The membrane was blotted with TEL antibody to confirm precipitation of 4/17 TEL-JAK2 (right). (E) Erk2 is activated in Ba/F3 cells expressing the TEL-JAK2 fusion variants. Ba/F3 cells expressing the TEL-JAK2 5/19 variant, ΔPNT mutant, or kinase-inactive (KI) mutant were starved of IL-3 for 4 h and treated with 100 μM PD98059 or dimethylsulfoxide for 10 min. Erk2 immunoprecipitated from these extracts was used for in vitro kinase assays using exogenous GST–Elk-1 as a substrate (top). Products were separated by SDS-PAGE followed by autoradiography. Parallel Erk2 Western blotting confirmed equal immunoprecipitation of the protein (bottom). Erk2 is also activated in cells expressing the 5/12 and 4/17 fusion variants (data not shown).

    Journal: Molecular and Cellular Biology

    Article Title: Socs-1 Inhibits TEL-JAK2-Mediated Transformation of Hematopoietic Cells through Inhibition of JAK2 Kinase Activity and Induction of Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.10.3547-3557.2001

    Figure Lengend Snippet: TEL-JAK2 associates with Shc and Grb2 and activates Erk2. (A) Shc is tyrosine phosphorylated in Ba/F3 cells expressing the TEL-JAK2 fusion variants and associates with TEL-JAK2. Extracts from Ba/F3 cells expressing the TEL-JAK2 fusion variants were immunoprecipitated (IP) with an anti-Shc antibody and blotted with 4G10 (top). TEL-JAK2 is seen as a doublet due to an alternative start site for translation in TEL. The membrane was blotted with a Shc antibody to confirm equal precipitation of the protein (bottom). Control experiments showed no evidence of association of Shc with inactive TEL-JAK2 mutants that lacked the PNT domain or were kinase inactive (data not shown). (B) Grb2 associates with the 5/19 and 5/12 TEL-JAK2 fusion variants. Extracts from panel A were immunoprecipitated with a Grb2 antibody and blotted with a TEL antibody. Association of Grb2 with the 4/17 variant was not detected. Control experiments showed no evidence of association of Grb2 with inactive TEL-JAK2 mutants that lacked the PNT domain or were kinase inactive (data not shown). The membrane was blotted with a Grb2 antibody to confirm equal precipitation (data not shown). (C) The SH2 domain of Grb2 directly associates with the 5/19 and 5/12 TEL-JAK2 fusion variants. Extracts from 293T cells expressing empty vector or the 5/19 TEL-JAK2 fusion variant were immunoprecipitated with a TEL antibody. Far-Western analysis was performed with purified GST and blotted with a GST antibody (left). Extracts from 293T cells expressing the 5/19 and 5/12 TEL-JAK2 fusion variants were immunoprecipitated with a TEL antibody, and far-Western analysis was performed with GST–Grb2 SH2 (middle). The membrane was blotted with TEL antibody to confirm precipitation of 5/19 and 5/12 TEL-JAK2 (right). (D) The SH2 domain of Grb2 does not directly associate with the 4/17 fusion variant. Extracts from 293T cells expressing the 4/17 TEL-JAK2 fusion variant was immunoprecipitated with a TEL antibody. Far-Western analysis was performed with purified GST (data not shown) or GST-Grb2 SH2 and blotted with a GST antibody (left). The membrane was blotted with TEL antibody to confirm precipitation of 4/17 TEL-JAK2 (right). (E) Erk2 is activated in Ba/F3 cells expressing the TEL-JAK2 fusion variants. Ba/F3 cells expressing the TEL-JAK2 5/19 variant, ΔPNT mutant, or kinase-inactive (KI) mutant were starved of IL-3 for 4 h and treated with 100 μM PD98059 or dimethylsulfoxide for 10 min. Erk2 immunoprecipitated from these extracts was used for in vitro kinase assays using exogenous GST–Elk-1 as a substrate (top). Products were separated by SDS-PAGE followed by autoradiography. Parallel Erk2 Western blotting confirmed equal immunoprecipitation of the protein (bottom). Erk2 is also activated in cells expressing the 5/12 and 4/17 fusion variants (data not shown).

    Article Snippet: Retroviral stocks were generated by transient cotransfection of 293T cells with the appropriate MSCV constructs together with a packaging construct (pIK6.1MCV.ecopac.UTd; Cell Genesys Inc., Foster City, Calif.) providing sequences necessary for retrovirus production ( ).

    Techniques: Expressing, Immunoprecipitation, Variant Assay, Plasmid Preparation, Western Blot, Purification, Mutagenesis, In Vitro, SDS Page, Autoradiography

    The SOCS box is required for proteasomal degradation of TEL-JAK2. (A) Titration of 5/19 TEL-JAK2 and Socs-1. Whole-cell extracts from 293T cells expressing decreasing amounts of 5/19 TEL-JAK2 and increasing amounts of Socs-1 were blotted with a TEL antibody. Western blotting of whole-cell extracts with a Myc antibody confirmed expression of Myc–Socs-1 (data not shown). (B) SOCS box of Socs-1 is required for decreased levels of TEL-JAK2 protein. Whole-cell extracts from 293T cells transfected with 2.5 μg of 5/19 TEL-JAK2 and increasing amounts of Myc–Socs-1 or Myc-dC40, which results in deletion of the SOCS box, were blotted with a TEL antibody (top). Western blotting of whole-cell extracts with a Myc antibody confirmed expression of Myc–Socs-1 and Myc-dC40 (bottom). (C) TEL-JAK2 in vitro kinase assay. 293T cells expressing 5/19 TEL-JAK2 with or without dC40 were starved of FBS for 4 h. Extracts from these cells were immunoprecipitated with a TEL antibody and used for in vitro kinase assays using GST-C'Gab2 as a substrate. Products were separated by SDS-PAGE followed by autoradiography (top). Parallel TEL immunoprecipitation and Western blotting confirmed equal immunoprecipitation of the protein (bottom). (D) Decrease in TEL-JAK2 protein level is mediated by proteasomal degradation. 293T cells transfected with 2.5 μg of 5/19 TEL-JAK2 and 10 μg Socs-1 or 10 μg dC40 were pulsed with [ 35 S]methionine and [ 35 S]cysteine for 15 min and chased for 2, 4, or 6 h. Indicated samples were treated with 25 μM lactacystin throughout the pulse and chase. Equivalent amount of extracts, as determined by the Bradford assay, were immunoprecipitated with a TEL antibody, separated by SDS-PAGE, and visualized by autoradiography. (E) Images from the pulse-chase were placed on a densitometer for analysis, and numerical results were plotted on a graph. Open diamonds represent degradation of TEL-JAK2 in cells expressing Jab, open triangles represent degradation of TEL-JAK2 in cells expressing dC40, and solid squares represent degradation of TEL-JAK2 in cells expressing Jab and treated with 25 μM lactacystin.

    Journal: Molecular and Cellular Biology

    Article Title: Socs-1 Inhibits TEL-JAK2-Mediated Transformation of Hematopoietic Cells through Inhibition of JAK2 Kinase Activity and Induction of Proteasome-Mediated Degradation

    doi: 10.1128/MCB.21.10.3547-3557.2001

    Figure Lengend Snippet: The SOCS box is required for proteasomal degradation of TEL-JAK2. (A) Titration of 5/19 TEL-JAK2 and Socs-1. Whole-cell extracts from 293T cells expressing decreasing amounts of 5/19 TEL-JAK2 and increasing amounts of Socs-1 were blotted with a TEL antibody. Western blotting of whole-cell extracts with a Myc antibody confirmed expression of Myc–Socs-1 (data not shown). (B) SOCS box of Socs-1 is required for decreased levels of TEL-JAK2 protein. Whole-cell extracts from 293T cells transfected with 2.5 μg of 5/19 TEL-JAK2 and increasing amounts of Myc–Socs-1 or Myc-dC40, which results in deletion of the SOCS box, were blotted with a TEL antibody (top). Western blotting of whole-cell extracts with a Myc antibody confirmed expression of Myc–Socs-1 and Myc-dC40 (bottom). (C) TEL-JAK2 in vitro kinase assay. 293T cells expressing 5/19 TEL-JAK2 with or without dC40 were starved of FBS for 4 h. Extracts from these cells were immunoprecipitated with a TEL antibody and used for in vitro kinase assays using GST-C'Gab2 as a substrate. Products were separated by SDS-PAGE followed by autoradiography (top). Parallel TEL immunoprecipitation and Western blotting confirmed equal immunoprecipitation of the protein (bottom). (D) Decrease in TEL-JAK2 protein level is mediated by proteasomal degradation. 293T cells transfected with 2.5 μg of 5/19 TEL-JAK2 and 10 μg Socs-1 or 10 μg dC40 were pulsed with [ 35 S]methionine and [ 35 S]cysteine for 15 min and chased for 2, 4, or 6 h. Indicated samples were treated with 25 μM lactacystin throughout the pulse and chase. Equivalent amount of extracts, as determined by the Bradford assay, were immunoprecipitated with a TEL antibody, separated by SDS-PAGE, and visualized by autoradiography. (E) Images from the pulse-chase were placed on a densitometer for analysis, and numerical results were plotted on a graph. Open diamonds represent degradation of TEL-JAK2 in cells expressing Jab, open triangles represent degradation of TEL-JAK2 in cells expressing dC40, and solid squares represent degradation of TEL-JAK2 in cells expressing Jab and treated with 25 μM lactacystin.

    Article Snippet: Retroviral stocks were generated by transient cotransfection of 293T cells with the appropriate MSCV constructs together with a packaging construct (pIK6.1MCV.ecopac.UTd; Cell Genesys Inc., Foster City, Calif.) providing sequences necessary for retrovirus production ( ).

    Techniques: Titration, Expressing, Western Blot, Transfection, In Vitro, Kinase Assay, Immunoprecipitation, SDS Page, Autoradiography, Bradford Assay, Pulse Chase

    Exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles. (A) BE-KO1 cells infected with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA1 (A1), ApoA2 (A2), ApoC1 (C1), ApoC2 (C2), ApoC3 (C3) and ApoH (H) and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. (B) ApoA1, ApoA2, ApoC1, ApoC2, ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with HCVcc at an MOI of 1. Expression of the apolipoproteins was determined by immunoblot analysis (upper), and infectious titers in the culture supernatants were determined at 72 h post-infection by focus-forming assay (lower). (C) Extracellular and intracellular HCV RNA in BE-KO1 cells expressing apolipoproteins and infected with HCVcc were determined at 72 h post-infection by qRT-PCR. (D) Specific infectivity was calculated as extracellular infectious titers/extracellular HCV RNA copies in BE-KO1 cells expressing apolipoproteins at 72 h post-infection. (E) 293T cells stably expressing CLDN1 and miR-122 (293T-CLDN/miR-122 cells) were infected with the lentiviral vectors, and the expressions of the apolipoproteins were determined by immunoblot analysis (upper). These cells were infected with HCVcc at an MOI of 1, and infectious titers in the supernatants were determined at 72 h post-infection by focus-forming assay (lower). In all cases, asterisks indicate significant differences (*, P

    Journal: PLoS Pathogens

    Article Title: Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

    doi: 10.1371/journal.ppat.1004534

    Figure Lengend Snippet: Exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles. (A) BE-KO1 cells infected with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA1 (A1), ApoA2 (A2), ApoC1 (C1), ApoC2 (C2), ApoC3 (C3) and ApoH (H) and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. (B) ApoA1, ApoA2, ApoC1, ApoC2, ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with HCVcc at an MOI of 1. Expression of the apolipoproteins was determined by immunoblot analysis (upper), and infectious titers in the culture supernatants were determined at 72 h post-infection by focus-forming assay (lower). (C) Extracellular and intracellular HCV RNA in BE-KO1 cells expressing apolipoproteins and infected with HCVcc were determined at 72 h post-infection by qRT-PCR. (D) Specific infectivity was calculated as extracellular infectious titers/extracellular HCV RNA copies in BE-KO1 cells expressing apolipoproteins at 72 h post-infection. (E) 293T cells stably expressing CLDN1 and miR-122 (293T-CLDN/miR-122 cells) were infected with the lentiviral vectors, and the expressions of the apolipoproteins were determined by immunoblot analysis (upper). These cells were infected with HCVcc at an MOI of 1, and infectious titers in the supernatants were determined at 72 h post-infection by focus-forming assay (lower). In all cases, asterisks indicate significant differences (*, P

    Article Snippet: Lipofection and lentiviral gene transduction The lentiviral vectors and ViraPower Lentiviral Packaging Mix (Life Technologies) were co-transfected into 293T cells by Trans IT LT-1 (Mirus), and the supernatants were recovered at 48 h post-transfection.

    Techniques: Infection, Transfection, Focus Forming Assay, Expressing, Quantitative RT-PCR, Stable Transfection

    Albumin led to the reduction of intracellular superoxide anion levels induced by hydrogen peroxide in human kidney cells. (A) Representative image of intracellular superoxide anion levels as detected by dihydroethidium (DHE) staining in human embryonic kidney (HEK) 293FT cells after 6-h exposure to hydrogen peroxide (500 μM) and pretreatment with phosphate buffered saline (PBS), human serum albumin (HSA, 3.0 g/dL) or γ-globulin (3.0 g/dL). As a control (CTR), we used cells that were not exposed to hydrogen peroxide. (B) The DHE fluorescent intensity per cell was quantified. HEK 293 FT cells were exposed to 500 μM hydrogen peroxide for 6 h with PBS, HSA, or γ-globulin. As a CTR, we used cells that were not exposed to hydrogen peroxide. Fluorescent intensities per cell were measured using ImageJ software. Data are presented as the mean ± standard error. (n = 8 imaged fields for each condition). * P

    Journal: PLoS ONE

    Article Title: Association between serum albumin level and incidence of end-stage renal disease in patients with Immunoglobulin A nephropathy: A possible role of albumin as an antioxidant agent

    doi: 10.1371/journal.pone.0196655

    Figure Lengend Snippet: Albumin led to the reduction of intracellular superoxide anion levels induced by hydrogen peroxide in human kidney cells. (A) Representative image of intracellular superoxide anion levels as detected by dihydroethidium (DHE) staining in human embryonic kidney (HEK) 293FT cells after 6-h exposure to hydrogen peroxide (500 μM) and pretreatment with phosphate buffered saline (PBS), human serum albumin (HSA, 3.0 g/dL) or γ-globulin (3.0 g/dL). As a control (CTR), we used cells that were not exposed to hydrogen peroxide. (B) The DHE fluorescent intensity per cell was quantified. HEK 293 FT cells were exposed to 500 μM hydrogen peroxide for 6 h with PBS, HSA, or γ-globulin. As a CTR, we used cells that were not exposed to hydrogen peroxide. Fluorescent intensities per cell were measured using ImageJ software. Data are presented as the mean ± standard error. (n = 8 imaged fields for each condition). * P

    Article Snippet: For HEK 293 cells, human serum albumin (HSA; A9731, Sigma-Aldrich) was used instead of BSA.

    Techniques: Staining, Software

    Knockdown of human GTPBP3 results in mitochondrial dysfunction. HEK-293 cells were transfected with GTPBP3 siRNAs or with negative control siRNAs, and oxygen consumption (A), superoxide detection in mitochondria with MitoSOX (B), mitochondrial membrane

    Journal:

    Article Title: Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification ▿Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification ▿ †

    doi: 10.1128/MCB.00946-08

    Figure Lengend Snippet: Knockdown of human GTPBP3 results in mitochondrial dysfunction. HEK-293 cells were transfected with GTPBP3 siRNAs or with negative control siRNAs, and oxygen consumption (A), superoxide detection in mitochondria with MitoSOX (B), mitochondrial membrane

    Article Snippet: Human HEK-293 cells were grown in minimum essential medium (Sigma).

    Techniques: Transfection, Negative Control

    Expression of GTPBP3 in HEK-293 cells. (A) Immunoblotting using anti-GTPBP3 of extracts from HEK-293 cells nontransfected (lanes 3, 5, and 7) or permanently transfected with plasmid pIC1055 (lanes 4, 6, and 8) or from E. coli strain DH5α/pIC1296

    Journal:

    Article Title: Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification ▿Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification ▿ †

    doi: 10.1128/MCB.00946-08

    Figure Lengend Snippet: Expression of GTPBP3 in HEK-293 cells. (A) Immunoblotting using anti-GTPBP3 of extracts from HEK-293 cells nontransfected (lanes 3, 5, and 7) or permanently transfected with plasmid pIC1055 (lanes 4, 6, and 8) or from E. coli strain DH5α/pIC1296

    Article Snippet: Human HEK-293 cells were grown in minimum essential medium (Sigma).

    Techniques: Expressing, Transfection, Plasmid Preparation

    Effect of GTPBP3 silencing on intracellular protein degradation. HEK-293 cells, untreated or treated as indicated, were incubated for 1 h with [ 3 H]leucine in the absence (A) or in the presence (B) of cycloheximide (200 μg/ml), and protein degradation

    Journal:

    Article Title: Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification ▿Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification ▿ †

    doi: 10.1128/MCB.00946-08

    Figure Lengend Snippet: Effect of GTPBP3 silencing on intracellular protein degradation. HEK-293 cells, untreated or treated as indicated, were incubated for 1 h with [ 3 H]leucine in the absence (A) or in the presence (B) of cycloheximide (200 μg/ml), and protein degradation

    Article Snippet: Human HEK-293 cells were grown in minimum essential medium (Sigma).

    Techniques: Incubation

    Lentiviral-mediated gene delivery to the mouse small intestine . a Schematic of virus production in 293T cells and mouse injection. Mice were injected at postnatal day 1 and sacrificed for analysis at later time points. b Green fluorescent protein (GFP) gene expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in the gastrointestinal tract and other organs. GFP expression is normalized to glyceraldehyde phosphate dehydrogenase expression and presented relative to levels in the stomach. Graph displays triplicate samples from representative animals. Data displayed as mean ± std. c GFP expression in isolated intestinal crypt and villus epithelium and mesenchymal cells by qRT-PCR. Graph displays triplicate samples from representative animal. Data displayed as mean ± std. d Hematoxylin and eosin stained normal intestinal crypt-villus unit. Solid line indicates apical epithelial border; dashed line indicates epithelial-mesenchymal border. e–g Co-labeling of DsRed-injected intestine with antibodies to GFP ( green ) and DsRed ( red ). Yellow box in e and f is enlarged in g. Arrowheads indicate dual expressing mesenchymal cells. Dashed line indicates epithelial-mesenchymal border. Solid white line in g marks the apical border. Bar = 25 μm.

    Journal: Biological Procedures Online

    Article Title: Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

    doi: 10.1007/s12575-009-9014-z

    Figure Lengend Snippet: Lentiviral-mediated gene delivery to the mouse small intestine . a Schematic of virus production in 293T cells and mouse injection. Mice were injected at postnatal day 1 and sacrificed for analysis at later time points. b Green fluorescent protein (GFP) gene expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in the gastrointestinal tract and other organs. GFP expression is normalized to glyceraldehyde phosphate dehydrogenase expression and presented relative to levels in the stomach. Graph displays triplicate samples from representative animals. Data displayed as mean ± std. c GFP expression in isolated intestinal crypt and villus epithelium and mesenchymal cells by qRT-PCR. Graph displays triplicate samples from representative animal. Data displayed as mean ± std. d Hematoxylin and eosin stained normal intestinal crypt-villus unit. Solid line indicates apical epithelial border; dashed line indicates epithelial-mesenchymal border. e–g Co-labeling of DsRed-injected intestine with antibodies to GFP ( green ) and DsRed ( red ). Yellow box in e and f is enlarged in g. Arrowheads indicate dual expressing mesenchymal cells. Dashed line indicates epithelial-mesenchymal border. Solid white line in g marks the apical border. Bar = 25 μm.

    Article Snippet: High-titer lentiviruses were produced in 293T cells by FuGene 6-mediated transient co-transfection (Roche Applied Science) of each of the four vectors.

    Techniques: Injection, Mouse Assay, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Staining, Labeling

    Overexpression of p53 by constructing lentivirus. The protein CDS region of murine p53 gene was inserted into the lentiviral expression vector pLVX-puro. Lentivirus encoding p53 (pLVX-p53) and control virus (pLVX) were packaged in 293T cells and used to infect MC3T3-E1 cells. We evaluated (A) mRNA levels of p53 with qPCR and (B) protein levels of p53 with western blotting, at 48 h after viral infection. ***P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Echinacoside suppresses dexamethasone-induced growth inhibition and apoptosis in osteoblastic MC3T3-E1 cells

    doi: 10.3892/etm.2018.6199

    Figure Lengend Snippet: Overexpression of p53 by constructing lentivirus. The protein CDS region of murine p53 gene was inserted into the lentiviral expression vector pLVX-puro. Lentivirus encoding p53 (pLVX-p53) and control virus (pLVX) were packaged in 293T cells and used to infect MC3T3-E1 cells. We evaluated (A) mRNA levels of p53 with qPCR and (B) protein levels of p53 with western blotting, at 48 h after viral infection. ***P

    Article Snippet: 293T cells (Shanghai GeneChem Co., Ltd., Shanghai, China) were transfected with a mixture of plasmids, including viral packaging plasmids and p53 expression plasmid (pLVX-p53) or control plasmid (pLVX) via Lipofectamine 2000 (Invitrogen: Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) based on the manufacturer's instructions.

    Techniques: Over Expression, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Infection

    LPS promotes RIP140 degradation through SOCS1-Rbx1 E3 ligase-mediated K48 polyubiquitination. (a) Immunoblot analysis of RIP140 and polyubiquitinated RIP140 in Raw264.7 macrophages stimulated for 6 h with indicated treatments. (b) Immunoblot analysis of RIP140, Rbx1 and actin in primary peritoneal macrophages stimulated for 24 h with a control treatment or LPS after transfection with control or Rbx1 siRNAs. (c) Immunoblot analysis of RIP140, SOCS1 and actin in primary peritoneal macrophages stimulated for 24 h with a control treatment or LPS after trasfection with control or SOCS1 siRNAs. (d, e, f) In vitro ubiqtuination analysis of RIP140 in 293T cells. 293T cells were transfected with expression plasmids as indicated and treated with 10 μM MG132 for 6 h before lysis. (d) Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-Rbx1. (e) Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-SOCS1. (f) 293T cells were transfected with Flag-tagged RIP140 and HA-tagged Ub in conjunction with HA-tagged SOCS1 wild type (Wt) or HA-tagged SOCS1-ΔSH (ΔSH). Cells were treated with 10 μM MG132 for 6 h before lysis. Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-SOCS1. All immunoblots were performed at least twice.

    Journal: Nature immunology

    Article Title: NF-?B-mediated degradation of the co-activator RIP140 regulates inflammatory response and contributes to endotoxin tolerance

    doi: 10.1038/ni.2238

    Figure Lengend Snippet: LPS promotes RIP140 degradation through SOCS1-Rbx1 E3 ligase-mediated K48 polyubiquitination. (a) Immunoblot analysis of RIP140 and polyubiquitinated RIP140 in Raw264.7 macrophages stimulated for 6 h with indicated treatments. (b) Immunoblot analysis of RIP140, Rbx1 and actin in primary peritoneal macrophages stimulated for 24 h with a control treatment or LPS after transfection with control or Rbx1 siRNAs. (c) Immunoblot analysis of RIP140, SOCS1 and actin in primary peritoneal macrophages stimulated for 24 h with a control treatment or LPS after trasfection with control or SOCS1 siRNAs. (d, e, f) In vitro ubiqtuination analysis of RIP140 in 293T cells. 293T cells were transfected with expression plasmids as indicated and treated with 10 μM MG132 for 6 h before lysis. (d) Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-Rbx1. (e) Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-SOCS1. (f) 293T cells were transfected with Flag-tagged RIP140 and HA-tagged Ub in conjunction with HA-tagged SOCS1 wild type (Wt) or HA-tagged SOCS1-ΔSH (ΔSH). Cells were treated with 10 μM MG132 for 6 h before lysis. Cell lysates were immunopreciptated with anti-Flag-agarose beads and immunoprecipitates were immunoblotted with anti-Flag to detect Flag-RIP140 or anti-HA to detect polyubiquitinated RIP140 and HA-SOCS1. All immunoblots were performed at least twice.

    Article Snippet: Briefly, 293T cells were transfected with expression vectors (hCD68 promoter-driven expression of wild type or mutant forms of RIP140) and packing system (System Biosciences).

    Techniques: Transfection, In Vitro, Expressing, Lysis, Western Blot

    Vascular endothelial growth factor C (VEGFC) is a novel target of miR-27b in colorectal cancer (CRC). (A) VEGFC is predicted as a novel target of miR-27b. (B) 293T cells were co-transfected with empty pmirGLO Dual-Luciferase reporter plasmids or VEGFC 3′UTR firefly luciferase reporter plasmids and pRL-TK-luciferase plasmids, together with miR-27b mimics or anti-miR-27b. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase. (C) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and expression of VEGFC was detected by western blotting. (D) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and VEGFC in culture medium was detected by ELISA. (E) VEGFC protein in xenografts from negative control (NC) and miR-27b mimics was detected by western blotting. Error bars represent the means ± SEM, * P

    Journal: PLoS ONE

    Article Title: miRNA-27b Targets Vascular Endothelial Growth Factor C to Inhibit Tumor Progression and Angiogenesis in Colorectal Cancer

    doi: 10.1371/journal.pone.0060687

    Figure Lengend Snippet: Vascular endothelial growth factor C (VEGFC) is a novel target of miR-27b in colorectal cancer (CRC). (A) VEGFC is predicted as a novel target of miR-27b. (B) 293T cells were co-transfected with empty pmirGLO Dual-Luciferase reporter plasmids or VEGFC 3′UTR firefly luciferase reporter plasmids and pRL-TK-luciferase plasmids, together with miR-27b mimics or anti-miR-27b. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase. (C) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and expression of VEGFC was detected by western blotting. (D) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and VEGFC in culture medium was detected by ELISA. (E) VEGFC protein in xenografts from negative control (NC) and miR-27b mimics was detected by western blotting. Error bars represent the means ± SEM, * P

    Article Snippet: The pRL-TK plasmids (Promega) were co-transfected into 293T cells with either the negative control mimic (5′-UUCUCCGAACGUGUCACGUTT-3′), miR-27b mimic (5′-UUCACAGUGGCUAAGUUCUGC-3′), or anti-miR-27b mimic (5′-GCAGAACUUAGCCACUGUGAA-3′) (GenePharma Tech, Shanghai, China) using Lipofectamine 2000 (Invitrogen).

    Techniques: Transfection, Luciferase, Activity Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Negative Control

    Promoter analysis of the sequence upstream of the human hnRNP K gene. A. Promoter luciferase reporter analysis in 293T, MCF-7 and HK2 cells. The relative promoter activities are represented as the fold increase versus expression from the activities of renilla plasmid. Values represent the mean ± SD of three independent experiments. T -test was used for statistical analysis. (a) P

    Journal: Biochimie

    Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

    doi: 10.1016/j.biochi.2014.12.002

    Figure Lengend Snippet: Promoter analysis of the sequence upstream of the human hnRNP K gene. A. Promoter luciferase reporter analysis in 293T, MCF-7 and HK2 cells. The relative promoter activities are represented as the fold increase versus expression from the activities of renilla plasmid. Values represent the mean ± SD of three independent experiments. T -test was used for statistical analysis. (a) P

    Article Snippet: Nuclear extracts were prepared from 293T cells using a nuclear extract kit (Sangon Biotech, China).

    Techniques: Sequencing, Luciferase, Expressing, Plasmid Preparation

    Analysis of the elements within K1 sequence of hnRNP K promoter. Various truncated genomic regions were introduced into the promoter-less pGL3-basic or pGL3-control vector, and the reporter constructs were transiently transfected into 293T (A), MCF-7 (B) or HK2 (C) cells. The relative promoter activities are represented as the fold increase versus expression from the activities of renilla plasmid. Values represent the mean ± SD of three independent experiments. T-test was used for statistical analysis. (a) P

    Journal: Biochimie

    Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

    doi: 10.1016/j.biochi.2014.12.002

    Figure Lengend Snippet: Analysis of the elements within K1 sequence of hnRNP K promoter. Various truncated genomic regions were introduced into the promoter-less pGL3-basic or pGL3-control vector, and the reporter constructs were transiently transfected into 293T (A), MCF-7 (B) or HK2 (C) cells. The relative promoter activities are represented as the fold increase versus expression from the activities of renilla plasmid. Values represent the mean ± SD of three independent experiments. T-test was used for statistical analysis. (a) P

    Article Snippet: Nuclear extracts were prepared from 293T cells using a nuclear extract kit (Sangon Biotech, China).

    Techniques: Sequencing, Plasmid Preparation, Construct, Transfection, Expressing

    Analysis of the transcription factors that could bind to hnRNP K promoter. A. In silico analysis showed that at least two Sp1 binding sites in hnRNP K promoter. B. ChIP analysis verified the binding of Sp1 with hnRNP K promoter in the 293T cells. There were two controls, a non-DNA negative control and an input DNA sample. PCR was performed using primers recognizing the K1 and K2 sequences in the hnRNP K promoter and the amplified products were separated in agarose gel and visualized with EB. The sizes of the PCR products were 513 bp (K1) and 229 bp (K2). Similar results were observed in three independent experiments. C. EMSA confirmed the exact binding site of Sp1 in the 37 bp probe. N.E. denotes nuclear extract of 293T cells. Cold means cold competitor and mutant means mutant competitor. D. Promoter luciferase reporter analysis of the sequences with mutant Sp1 binding sites in 293T and HK2 cells. Values represent the mean ± SD of three independent experiments. T-test was used for statistical analysis. (a) P

    Journal: Biochimie

    Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

    doi: 10.1016/j.biochi.2014.12.002

    Figure Lengend Snippet: Analysis of the transcription factors that could bind to hnRNP K promoter. A. In silico analysis showed that at least two Sp1 binding sites in hnRNP K promoter. B. ChIP analysis verified the binding of Sp1 with hnRNP K promoter in the 293T cells. There were two controls, a non-DNA negative control and an input DNA sample. PCR was performed using primers recognizing the K1 and K2 sequences in the hnRNP K promoter and the amplified products were separated in agarose gel and visualized with EB. The sizes of the PCR products were 513 bp (K1) and 229 bp (K2). Similar results were observed in three independent experiments. C. EMSA confirmed the exact binding site of Sp1 in the 37 bp probe. N.E. denotes nuclear extract of 293T cells. Cold means cold competitor and mutant means mutant competitor. D. Promoter luciferase reporter analysis of the sequences with mutant Sp1 binding sites in 293T and HK2 cells. Values represent the mean ± SD of three independent experiments. T-test was used for statistical analysis. (a) P

    Article Snippet: Nuclear extracts were prepared from 293T cells using a nuclear extract kit (Sangon Biotech, China).

    Techniques: In Silico, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Mutagenesis, Luciferase

    T cell multiepitopic B (TMEP-B) design, construction of pcDNA-TMEP-B plasmid, and TMEP-B expression analysis by Western blot. ( A ) Scheme of TMEP-B protein. ( B ) Map of the plasmid pcDNA-TMEP-B. ( C ) Expression of TMEP-B construct by Western blot. The 293T cells were mock-infected or infected with 5 pfu/cell of Western Reserve (WR) or vaccinia virus (VACV) that expresses the T7 RNA polymerase (VT7) viruses, and transfected 1 h later with 5 μg of pcDNA-TMEP-B or pMax-GFP. At 6 h post-infection, cells were harvested and lysed in Laemmli buffer with mercapoethanol and cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody to evaluate TMEP-B expression.

    Journal: Viruses

    Article Title: Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B

    doi: 10.3390/v10080424

    Figure Lengend Snippet: T cell multiepitopic B (TMEP-B) design, construction of pcDNA-TMEP-B plasmid, and TMEP-B expression analysis by Western blot. ( A ) Scheme of TMEP-B protein. ( B ) Map of the plasmid pcDNA-TMEP-B. ( C ) Expression of TMEP-B construct by Western blot. The 293T cells were mock-infected or infected with 5 pfu/cell of Western Reserve (WR) or vaccinia virus (VACV) that expresses the T7 RNA polymerase (VT7) viruses, and transfected 1 h later with 5 μg of pcDNA-TMEP-B or pMax-GFP. At 6 h post-infection, cells were harvested and lysed in Laemmli buffer with mercapoethanol and cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody to evaluate TMEP-B expression.

    Article Snippet: Transfection Assay and Expression of TMEP-B Protein by Western Blot Analysis To determine the correct expression of TMEP-B protein from DNA-TMEP vector, 293T cells (1 × 106 ) were mock-infected or infected with 5 pfu/cell of WR or VT7 viruses and transfected 1 h later with 5 μg of DNA-TMEP or pMax-GFP (Lonza, Basel, Switzerland) using Lipofectamine 2000 (Invitrogen) according to manufacturer’s recommendations.

    Techniques: Plasmid Preparation, Expressing, Western Blot, Construct, Infection, Transfection, SDS Page

    Structure of sgp130-E10 and its interaction with Hyper-IL-6. A , schematic overview of sgp130-E10 in complex with IL-6/IL-6R. B , immunoprecipitation with conditioned medium from HEK-293 cell cultures transiently transfected with plasmids coding for sgp130-E10Myc-His

    Journal: The Journal of Biological Chemistry

    Article Title: Alternative Intronic Polyadenylation Generates the Interleukin-6 Trans-signaling Inhibitor sgp130-E10 *

    doi: 10.1074/jbc.M114.560938

    Figure Lengend Snippet: Structure of sgp130-E10 and its interaction with Hyper-IL-6. A , schematic overview of sgp130-E10 in complex with IL-6/IL-6R. B , immunoprecipitation with conditioned medium from HEK-293 cell cultures transiently transfected with plasmids coding for sgp130-E10Myc-His

    Article Snippet: CHO-K1 cells and HEK-293 cells were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen; Braunschweig, Germany).

    Techniques: Immunoprecipitation, Transfection

    Current-voltage relationship of SARS-CoV E protein and inhibition by HMA. (A) Example traces of current flowing through cells expressing SARS-CoV E protein, vector alone-transfected cells and untransfected HEK-293 cells. The cells were held at 0 mV and stepped to various potentials from −100 to 70 mV (in steps of 10 mV). (B) Whole-cell I–V curve in which peak current amplitudes were plotted against test potentials. Notice the significant large inward and outward currents recorded from SARS-CoV E protein, in contrast to vector alone and untransfected HEK-293 cell controls (*, two-tail unpaired T test, p

    Journal: PLoS Pathogens

    Article Title: Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

    doi: 10.1371/journal.ppat.1000511

    Figure Lengend Snippet: Current-voltage relationship of SARS-CoV E protein and inhibition by HMA. (A) Example traces of current flowing through cells expressing SARS-CoV E protein, vector alone-transfected cells and untransfected HEK-293 cells. The cells were held at 0 mV and stepped to various potentials from −100 to 70 mV (in steps of 10 mV). (B) Whole-cell I–V curve in which peak current amplitudes were plotted against test potentials. Notice the significant large inward and outward currents recorded from SARS-CoV E protein, in contrast to vector alone and untransfected HEK-293 cell controls (*, two-tail unpaired T test, p

    Article Snippet: SARS-CoV E construct and transient expression of SARS-CoV E cDNA in HEK-293 cells The full-length SARS-CoV E protein gene was cloned into pIRES-AcGFP1 (Clonetech) vector by using the restriction enzymes BglII and PstI.

    Techniques: Inhibition, Expressing, Plasmid Preparation, Transfection

    Effect of LR1-C1 mutations on affinity for selected bNAbs. Virus stocks were obtained from transfected 293T cells. Increase in Ab binding was determined by comparison of the virus captured by different monoclonal antibodies and quantified by p24 when similar amounts of virus (AC10_29 in red and filled circles and LR1-C1 in blue and filled squares) were used as input in a Virion Capture Assay (VCA) with the Abs 4E10 (MPER), 2F5 (MPER), 447-52D (V3), VRC01 (CD4bs), 2G12 (N332 V3 glycan patch), b12 (CD4bs), PG16 (quaternary; V1-V1 glycan apex), PGT151 (quaternary; gp120-gp41 integrase) and 5F3 (gp41). Similar amounts of virus suspension with no mAb were used as controls for both viruses (AC10_29 in grey and filled circles and LR1-C1 in grey and filled squares). Statistical analysis was conducted by R using One-way NOVA followed by Newman Keuls post hoc test at each input concentration (***p

    Journal: PLoS ONE

    Article Title: Guiding the humoral response against HIV-1 toward a MPER adjacent region by immunization with a VLP-formulated antibody-selected envelope variant

    doi: 10.1371/journal.pone.0208345

    Figure Lengend Snippet: Effect of LR1-C1 mutations on affinity for selected bNAbs. Virus stocks were obtained from transfected 293T cells. Increase in Ab binding was determined by comparison of the virus captured by different monoclonal antibodies and quantified by p24 when similar amounts of virus (AC10_29 in red and filled circles and LR1-C1 in blue and filled squares) were used as input in a Virion Capture Assay (VCA) with the Abs 4E10 (MPER), 2F5 (MPER), 447-52D (V3), VRC01 (CD4bs), 2G12 (N332 V3 glycan patch), b12 (CD4bs), PG16 (quaternary; V1-V1 glycan apex), PGT151 (quaternary; gp120-gp41 integrase) and 5F3 (gp41). Similar amounts of virus suspension with no mAb were used as controls for both viruses (AC10_29 in grey and filled circles and LR1-C1 in grey and filled squares). Statistical analysis was conducted by R using One-way NOVA followed by Newman Keuls post hoc test at each input concentration (***p

    Article Snippet: Env expression in 293T cells was also analysed by western blotting after lysing with a Cell lysis buffer (Cell Signaling Tecnology, Danvers, MA, U.S.A) and boiled for 10 minutes.

    Techniques: Transfection, Binding Assay, Concentration Assay

    Higher expression of specific genes in cells expressing S547A mutated p65. qRT PCR analysis of IL8 ( A ), A20 ( B ), Sele ( C ), VCAM1 ( D ), CXCL1 ( E ) and CD83 ( F ) mRNA level after 4 and 8 hours of etoposide treatment in HEK-293 cells expressing either p65 wt or p65 S547A .

    Journal: PLoS ONE

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    doi: 10.1371/journal.pone.0038246

    Figure Lengend Snippet: Higher expression of specific genes in cells expressing S547A mutated p65. qRT PCR analysis of IL8 ( A ), A20 ( B ), Sele ( C ), VCAM1 ( D ), CXCL1 ( E ) and CD83 ( F ) mRNA level after 4 and 8 hours of etoposide treatment in HEK-293 cells expressing either p65 wt or p65 S547A .

    Article Snippet: HEK-293 cells were next infected with the lentiviral stock and treated with Blasticidin (Invivogen) to select stably transduced HEK-293 cells expressing siRNA resistant HA-p65.

    Techniques: Expressing, Quantitative RT-PCR

    Higher IL8 protein level in cells expressing S547A mutated p65. ELISA analysis of IL8 protein level after the indicated times of etoposide treatment in HEK-293 cells expressing either p65 wt or p65 S547A .

    Journal: PLoS ONE

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    doi: 10.1371/journal.pone.0038246

    Figure Lengend Snippet: Higher IL8 protein level in cells expressing S547A mutated p65. ELISA analysis of IL8 protein level after the indicated times of etoposide treatment in HEK-293 cells expressing either p65 wt or p65 S547A .

    Article Snippet: HEK-293 cells were next infected with the lentiviral stock and treated with Blasticidin (Invivogen) to select stably transduced HEK-293 cells expressing siRNA resistant HA-p65.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    p65 interaction with ATM and p65 Ser 547 phosphorylation by ATM. ( A ) Confirmation of a direct interaction between ATM and p65 . Bacterially expressed GST or GST-ATM (1–247) fusion protein were purified on glutathione agarose beads and used to pull-down p65 from HEK-293 over-expressing HA-p65 cell lysate. Pulled down p65 was detected by immunoblotting with a p65 antibody (upper panel) and GST proteins were stained with coomassie bleu on the PVDF membrane. ( B ) Identification of the ATM target residue . Schematic representation of the different GST-p65 substrates used in the kinase assay. ( C ) In vitro kinase assay . Immunoprecipitated ATM from HEK-293 cells was incubated with GST-p53 and different GST-p65 proteins in presence of [γ− 32 P]ATP. The radiolabelled bands (upper panels) represent auto-phosphorylated ATM, phosphorylated GST-p53 or GST-p65. Levels of ATM and of substrate present in each reaction were determined by western blotting and by coomassie blue staining respectively (lower panels). ( D ) As in (C) In vitro kinase assay but with wt or mutated GST-p53 and GST-p65 proteins as substrates. ATM inhibitor KU55933 was added in some reaction samples as indicated. The same detection methodology than in ( C ) was used. ( E ) As in (D) in vitro kinase assay, but with purified recombinant ATM wt or kd instead of immunoprecipitated ATM and KU55933 utilization. ( F ) Conservation of Ser 547 among different species. Alignment of p65 C-terminal sequence from different mammalian and bird species.

    Journal: PLoS ONE

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    doi: 10.1371/journal.pone.0038246

    Figure Lengend Snippet: p65 interaction with ATM and p65 Ser 547 phosphorylation by ATM. ( A ) Confirmation of a direct interaction between ATM and p65 . Bacterially expressed GST or GST-ATM (1–247) fusion protein were purified on glutathione agarose beads and used to pull-down p65 from HEK-293 over-expressing HA-p65 cell lysate. Pulled down p65 was detected by immunoblotting with a p65 antibody (upper panel) and GST proteins were stained with coomassie bleu on the PVDF membrane. ( B ) Identification of the ATM target residue . Schematic representation of the different GST-p65 substrates used in the kinase assay. ( C ) In vitro kinase assay . Immunoprecipitated ATM from HEK-293 cells was incubated with GST-p53 and different GST-p65 proteins in presence of [γ− 32 P]ATP. The radiolabelled bands (upper panels) represent auto-phosphorylated ATM, phosphorylated GST-p53 or GST-p65. Levels of ATM and of substrate present in each reaction were determined by western blotting and by coomassie blue staining respectively (lower panels). ( D ) As in (C) In vitro kinase assay but with wt or mutated GST-p53 and GST-p65 proteins as substrates. ATM inhibitor KU55933 was added in some reaction samples as indicated. The same detection methodology than in ( C ) was used. ( E ) As in (D) in vitro kinase assay, but with purified recombinant ATM wt or kd instead of immunoprecipitated ATM and KU55933 utilization. ( F ) Conservation of Ser 547 among different species. Alignment of p65 C-terminal sequence from different mammalian and bird species.

    Article Snippet: HEK-293 cells were next infected with the lentiviral stock and treated with Blasticidin (Invivogen) to select stably transduced HEK-293 cells expressing siRNA resistant HA-p65.

    Techniques: Purification, Expressing, Staining, Kinase Assay, In Vitro, Immunoprecipitation, Incubation, Western Blot, Recombinant, Sequencing

    No impact of p65 Ser 547 phosphorylation on global NF-κB transcriptional activity. ( A ) HEK-293 cells were transfected with κB Luciferase plasmid and with increasing amount of either wt or S547A mutated p65. Twenty-four hours later the cells were treated for 8 h with etoposide or left untreated. NT, non transfected.

    Journal: PLoS ONE

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    doi: 10.1371/journal.pone.0038246

    Figure Lengend Snippet: No impact of p65 Ser 547 phosphorylation on global NF-κB transcriptional activity. ( A ) HEK-293 cells were transfected with κB Luciferase plasmid and with increasing amount of either wt or S547A mutated p65. Twenty-four hours later the cells were treated for 8 h with etoposide or left untreated. NT, non transfected.

    Article Snippet: HEK-293 cells were next infected with the lentiviral stock and treated with Blasticidin (Invivogen) to select stably transduced HEK-293 cells expressing siRNA resistant HA-p65.

    Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation

    Creation of HEK-293 cell lines expressing either wt or S547A mutated p65. ( A ) Schematic representation of the 2 silent mutations inserted in the sequence of p65 within the sequence targeted by p65 siRNA, conferring to exogenous HA-p65 the resistance to the siRNA. ( B ) Western blot analysis of cytoplasmic and nuclear p65 protein (p65 antibody) and of exogenous p65 (HA antibody) in non-treated or etoposide treated HEK-293 and HEK-293 stably expressing sip65 resistant HA-p65. All cell lines were either transfected with control or p65 siRNA. ( C–D ) Comparison of NF-κB activation after etoposide treatment, by (C) EMSA and by (D) qRT PCR of IκBα mRNA, between HEK-293 and HEK-293 stably expressing sip65 resistant HA-p65. All cell lines were transfected either with control or p65 siRNA. ( C ) NF-κB DNA binding was analyzed using a probe corresponding to the HIV-1 long terminal repeat κB site (top panel). The presence of p65 and p50 protein in the binding complex observed in HEK-293 was analyzed by supershift with p65 and p50 antibodies (bottom panel). ns, non specific band.

    Journal: PLoS ONE

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    doi: 10.1371/journal.pone.0038246

    Figure Lengend Snippet: Creation of HEK-293 cell lines expressing either wt or S547A mutated p65. ( A ) Schematic representation of the 2 silent mutations inserted in the sequence of p65 within the sequence targeted by p65 siRNA, conferring to exogenous HA-p65 the resistance to the siRNA. ( B ) Western blot analysis of cytoplasmic and nuclear p65 protein (p65 antibody) and of exogenous p65 (HA antibody) in non-treated or etoposide treated HEK-293 and HEK-293 stably expressing sip65 resistant HA-p65. All cell lines were either transfected with control or p65 siRNA. ( C–D ) Comparison of NF-κB activation after etoposide treatment, by (C) EMSA and by (D) qRT PCR of IκBα mRNA, between HEK-293 and HEK-293 stably expressing sip65 resistant HA-p65. All cell lines were transfected either with control or p65 siRNA. ( C ) NF-κB DNA binding was analyzed using a probe corresponding to the HIV-1 long terminal repeat κB site (top panel). The presence of p65 and p50 protein in the binding complex observed in HEK-293 was analyzed by supershift with p65 and p50 antibodies (bottom panel). ns, non specific band.

    Article Snippet: HEK-293 cells were next infected with the lentiviral stock and treated with Blasticidin (Invivogen) to select stably transduced HEK-293 cells expressing siRNA resistant HA-p65.

    Techniques: Expressing, Sequencing, Western Blot, Stable Transfection, Transfection, Activation Assay, Quantitative RT-PCR, Binding Assay

    Ser 547 phosphorylation–dependent IL8 gene regulation mechanism involve HDAC. ( A–B ) HEK-293 expressing either p65 wt or p65 S547A were non-treated or treated 4 hours with etoposide in absence or presence of TSA, and mRNA levels of IL8 ( A ) and IκBα ( B ) were analyzed by qRT PCR. ( C–D ) Recruitments of Lys 9 acetylated histone 3 on IL8 ( C ) and IκBα ( D ) promoters were measured by ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated with etoposide for the indicated periods. ( E ) HA-tagged p65 wt or p65 S547A were immunoprecipitated from lysates of HEK-293 cell transfected with expression plasmid for these two proteins or non transfected as control. Co-immunoprecipitated HDAC1 protein was detected by western blotting (upper panel). Level of immunoprecipitated HA-p65 in the different samples was determined by western blotting (lower panel). A portion of whole cell extract was added on the gel as input. ( F ) Recruitment of HDAC1 on IL8 promoter was measured by ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated 5 hours with etoposide or left untreated. *, significantly different (P

    Journal: PLoS ONE

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    doi: 10.1371/journal.pone.0038246

    Figure Lengend Snippet: Ser 547 phosphorylation–dependent IL8 gene regulation mechanism involve HDAC. ( A–B ) HEK-293 expressing either p65 wt or p65 S547A were non-treated or treated 4 hours with etoposide in absence or presence of TSA, and mRNA levels of IL8 ( A ) and IκBα ( B ) were analyzed by qRT PCR. ( C–D ) Recruitments of Lys 9 acetylated histone 3 on IL8 ( C ) and IκBα ( D ) promoters were measured by ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated with etoposide for the indicated periods. ( E ) HA-tagged p65 wt or p65 S547A were immunoprecipitated from lysates of HEK-293 cell transfected with expression plasmid for these two proteins or non transfected as control. Co-immunoprecipitated HDAC1 protein was detected by western blotting (upper panel). Level of immunoprecipitated HA-p65 in the different samples was determined by western blotting (lower panel). A portion of whole cell extract was added on the gel as input. ( F ) Recruitment of HDAC1 on IL8 promoter was measured by ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated 5 hours with etoposide or left untreated. *, significantly different (P

    Article Snippet: HEK-293 cells were next infected with the lentiviral stock and treated with Blasticidin (Invivogen) to select stably transduced HEK-293 cells expressing siRNA resistant HA-p65.

    Techniques: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot

    Similar etoposide-induced gene expression profile in cells expressing wt and S547D mutated p65. qRT PCR analysis of IL8 (A), and IκBα (B) mRNA level after 4 and 8 hours of etoposide treatment in HEK-293 cells expressing either p65 wt , p65 S547A , or p65 S547D .

    Journal: PLoS ONE

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    doi: 10.1371/journal.pone.0038246

    Figure Lengend Snippet: Similar etoposide-induced gene expression profile in cells expressing wt and S547D mutated p65. qRT PCR analysis of IL8 (A), and IκBα (B) mRNA level after 4 and 8 hours of etoposide treatment in HEK-293 cells expressing either p65 wt , p65 S547A , or p65 S547D .

    Article Snippet: HEK-293 cells were next infected with the lentiviral stock and treated with Blasticidin (Invivogen) to select stably transduced HEK-293 cells expressing siRNA resistant HA-p65.

    Techniques: Expressing, Quantitative RT-PCR

    Identical binding of wt and S547A mutated p65 to IL8 κB site. ( A ) NF-κB binding to IL8 promoter, in HEK-293 cells expressing either p65 wt or p65 S547A , non-treated or treated with etoposide, was analyzed by EMSA with a probe corresponding to the κB site of IL8 promoter. ( B ) The presence of p65 protein in the binding complex observed in ( A ) was analyzed by supershift with p65 antibody. ( C ) Recruitment of p65 on IL8 promoter was measured by p65 ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated with etoposide for the indicated periods. ns, non significantly different.

    Journal: PLoS ONE

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    doi: 10.1371/journal.pone.0038246

    Figure Lengend Snippet: Identical binding of wt and S547A mutated p65 to IL8 κB site. ( A ) NF-κB binding to IL8 promoter, in HEK-293 cells expressing either p65 wt or p65 S547A , non-treated or treated with etoposide, was analyzed by EMSA with a probe corresponding to the κB site of IL8 promoter. ( B ) The presence of p65 protein in the binding complex observed in ( A ) was analyzed by supershift with p65 antibody. ( C ) Recruitment of p65 on IL8 promoter was measured by p65 ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated with etoposide for the indicated periods. ns, non significantly different.

    Article Snippet: HEK-293 cells were next infected with the lentiviral stock and treated with Blasticidin (Invivogen) to select stably transduced HEK-293 cells expressing siRNA resistant HA-p65.

    Techniques: Binding Assay, Expressing, Chromatin Immunoprecipitation

    Targeted transduction and delivery of PSCA antigen gene into dendritic cells (DCs) by DCLV-PSCA. (A) 293T cells were transfected transiently with plasmids FUW-Null (mock control, blue line) or FUW-PSCA (red line). Two days later, cells were collected and stained for PSCA expression analyzed by flow cytometry. 293T cells stained with the isotype antibody were included as a control (grey shade area). (B) 293T cells were transfected transiently with plasmids FUW-PSCA, SVGmu, and other necessary lentiviral packaging plasmids to produce DCLV-PSCA vectors. Fresh virus supernatant was used to transduce 293T cells (blue line) or 293T.hDC-SIGN cells (red line) with MOI = 10. PSCA expression was analyzed by flow cytometry 3 days post-transduction. (C) Bone marrow-derived DCs were transduced with a mock vector DC-LV-Null or DC-LV-PSCA vector. Five days later, CD11c and PSCA expression were assessed by flow cytometric analysis. All experiments were repeated three times and the representative data is shown.

    Journal: PLoS ONE

    Article Title: Dendritic Cell-Directed Vaccination with a Lentivector Encoding PSCA for Prostate Cancer in Mice

    doi: 10.1371/journal.pone.0048866

    Figure Lengend Snippet: Targeted transduction and delivery of PSCA antigen gene into dendritic cells (DCs) by DCLV-PSCA. (A) 293T cells were transfected transiently with plasmids FUW-Null (mock control, blue line) or FUW-PSCA (red line). Two days later, cells were collected and stained for PSCA expression analyzed by flow cytometry. 293T cells stained with the isotype antibody were included as a control (grey shade area). (B) 293T cells were transfected transiently with plasmids FUW-PSCA, SVGmu, and other necessary lentiviral packaging plasmids to produce DCLV-PSCA vectors. Fresh virus supernatant was used to transduce 293T cells (blue line) or 293T.hDC-SIGN cells (red line) with MOI = 10. PSCA expression was analyzed by flow cytometry 3 days post-transduction. (C) Bone marrow-derived DCs were transduced with a mock vector DC-LV-Null or DC-LV-PSCA vector. Five days later, CD11c and PSCA expression were assessed by flow cytometric analysis. All experiments were repeated three times and the representative data is shown.

    Article Snippet: Briefly, 293T cells cultured in a 15-cm tissue culture plate (BD Biosciences, San Jose, CA, USA) were transfected via a standard calcium phosphate precipitation method with the following plasmids: the lentiviral backbone plasmid FUW-PSCA (37.5 µg, ), the plasmid encoding the mutant Sindbis virus glycoprotein (SVGmu, 18.75 µg, ), and the packaging plasmids (pMDLg/pRRE and pRSV-Rev, 18.75 µg each).

    Techniques: Transduction, Transfection, Staining, Expressing, Flow Cytometry, Cytometry, Derivative Assay, Plasmid Preparation

    PSCA-specific T cell response after a single dose of in vivo immunization with DCLV-PSCA. (A) Male C57BL/6 mice were immunized with 6×10 7 TU of DCLV-PSCA through different administration routes: intraperitoneal space (i.p.), subcutaneous area (s.c.), intramuscular area (i.m.), footpad (f.p.), or intradermal (the base of tail, i.d.). One immunization group was included as a negative control. Two weeks after immunization, splenocytes from mice were harvested and analyzed for the presence of PSCA-specific CD8 + T cells by restimulating splenocytes with a PSCA peptide (PSCA 83-91 ), followed by intracellular staining for IFN-γ and surface staining for CD8. Percentage of IFN-γ-secreting CD8 + T cells is indicated. (B) Statistical comparison of immunization elicited by administration of DCLV-PSCA among different administration routes. (C) Male C57BL/6 mice were immunized with different doses of DCLV-PSCA vectors (0, 2, 10, 40 and 80 million TU) at the base of tail. Two weeks post-vaccination, PSCA-specific CD8 + T cells from the spleen were analyzed by restimulating with the peptide PSCA 83-91 , followed by intracellular staining for IFN-γ. (D) Production of PSCA-specific IFN-γ-secreting cells from both spleen (SP) and inguinal lymph node (LN) was evaluated by restimulation with the PSCA 83-91 peptide, followed by ELISPOT analysis for IFN-γ. (E) Production of PSCA-specific IL-2 from splenocytes (with CD8 + T cells depleted) was measured by restimulation with 293T cell lysate transfected to express PSCA, followed by the ELISPOT analysis for IL-2. (**: P

    Journal: PLoS ONE

    Article Title: Dendritic Cell-Directed Vaccination with a Lentivector Encoding PSCA for Prostate Cancer in Mice

    doi: 10.1371/journal.pone.0048866

    Figure Lengend Snippet: PSCA-specific T cell response after a single dose of in vivo immunization with DCLV-PSCA. (A) Male C57BL/6 mice were immunized with 6×10 7 TU of DCLV-PSCA through different administration routes: intraperitoneal space (i.p.), subcutaneous area (s.c.), intramuscular area (i.m.), footpad (f.p.), or intradermal (the base of tail, i.d.). One immunization group was included as a negative control. Two weeks after immunization, splenocytes from mice were harvested and analyzed for the presence of PSCA-specific CD8 + T cells by restimulating splenocytes with a PSCA peptide (PSCA 83-91 ), followed by intracellular staining for IFN-γ and surface staining for CD8. Percentage of IFN-γ-secreting CD8 + T cells is indicated. (B) Statistical comparison of immunization elicited by administration of DCLV-PSCA among different administration routes. (C) Male C57BL/6 mice were immunized with different doses of DCLV-PSCA vectors (0, 2, 10, 40 and 80 million TU) at the base of tail. Two weeks post-vaccination, PSCA-specific CD8 + T cells from the spleen were analyzed by restimulating with the peptide PSCA 83-91 , followed by intracellular staining for IFN-γ. (D) Production of PSCA-specific IFN-γ-secreting cells from both spleen (SP) and inguinal lymph node (LN) was evaluated by restimulation with the PSCA 83-91 peptide, followed by ELISPOT analysis for IFN-γ. (E) Production of PSCA-specific IL-2 from splenocytes (with CD8 + T cells depleted) was measured by restimulation with 293T cell lysate transfected to express PSCA, followed by the ELISPOT analysis for IL-2. (**: P

    Article Snippet: Briefly, 293T cells cultured in a 15-cm tissue culture plate (BD Biosciences, San Jose, CA, USA) were transfected via a standard calcium phosphate precipitation method with the following plasmids: the lentiviral backbone plasmid FUW-PSCA (37.5 µg, ), the plasmid encoding the mutant Sindbis virus glycoprotein (SVGmu, 18.75 µg, ), and the packaging plasmids (pMDLg/pRRE and pRSV-Rev, 18.75 µg each).

    Techniques: In Vivo, Mouse Assay, Negative Control, Staining, Enzyme-linked Immunospot, Transfection

    Fluorescent recombineering reporter. A) Lentiviral vector pDual-eGFP Y203 was used to deliver a Yellow florescent protein variant of eGFP to serve as the recombineering target transgene. B) Fluorescence excitation and emission spectra of purified eGFP and eGFP Y203 proteins. C-F) Flow cytometry was used to quantify 293T-Green recombinant and 293T-Yellow parental phenotypes. C) Untransduced cells were quantified in the R1 gate, D) pDual-eGFP transduced Green cells were quantified in the P2 gate and E) pDual-eGFP Y203 transduced Yellow cells were quantified in the P3 gate. F) Example results from one recombination experiment using Yellow cells as a target and an oligo that codes for Green fluorescence.

    Journal: PLoS ONE

    Article Title: Herpes ICP8 protein stimulates homologous recombination in human cells

    doi: 10.1371/journal.pone.0200955

    Figure Lengend Snippet: Fluorescent recombineering reporter. A) Lentiviral vector pDual-eGFP Y203 was used to deliver a Yellow florescent protein variant of eGFP to serve as the recombineering target transgene. B) Fluorescence excitation and emission spectra of purified eGFP and eGFP Y203 proteins. C-F) Flow cytometry was used to quantify 293T-Green recombinant and 293T-Yellow parental phenotypes. C) Untransduced cells were quantified in the R1 gate, D) pDual-eGFP transduced Green cells were quantified in the P2 gate and E) pDual-eGFP Y203 transduced Yellow cells were quantified in the P3 gate. F) Example results from one recombination experiment using Yellow cells as a target and an oligo that codes for Green fluorescence.

    Article Snippet: 293T cells transfected with lentiviral vectors in the presence and absence of doxycycline (1 μg/ml) were collected 24 hours after transfection and analysed by Western blot as described by Abcam.

    Techniques: Plasmid Preparation, Variant Assay, Fluorescence, Purification, Flow Cytometry, Cytometry, Recombinant

    Optimization of human cell recombineering. During the course of developing this platform for human cell recombineering by ICP8, the frequency of gene targeting varied by 10 to 20 fold, influenced by the design and concentration of the oligo substrate, as well as the cell density in the well during oligo transfection. A) ICP8 and endogenous recombination was significantly improved by longer and antisense oligos . 293T-Yellow cells were sequentially transfected with pCMV-ICP8 and with oligos that donate the sequence for converting Yellow to Green fluorescence. Recombination was measured as a function of oligo size and the targeted strand. AS oligo indicates that the oligo corresponds to the antisense strand of eGFP Y203 and S oligo indicates that the oligo corresponds to the sense strand of eGFP Y203 . The no oligo background for 293T or 293T-pCMV-ICP8 was subtracted from each data point and never exceeded 0.004%. Fisher’s Exact Test was used to evaluate statistical significance with **** indicating P

    Journal: PLoS ONE

    Article Title: Herpes ICP8 protein stimulates homologous recombination in human cells

    doi: 10.1371/journal.pone.0200955

    Figure Lengend Snippet: Optimization of human cell recombineering. During the course of developing this platform for human cell recombineering by ICP8, the frequency of gene targeting varied by 10 to 20 fold, influenced by the design and concentration of the oligo substrate, as well as the cell density in the well during oligo transfection. A) ICP8 and endogenous recombination was significantly improved by longer and antisense oligos . 293T-Yellow cells were sequentially transfected with pCMV-ICP8 and with oligos that donate the sequence for converting Yellow to Green fluorescence. Recombination was measured as a function of oligo size and the targeted strand. AS oligo indicates that the oligo corresponds to the antisense strand of eGFP Y203 and S oligo indicates that the oligo corresponds to the sense strand of eGFP Y203 . The no oligo background for 293T or 293T-pCMV-ICP8 was subtracted from each data point and never exceeded 0.004%. Fisher’s Exact Test was used to evaluate statistical significance with **** indicating P

    Article Snippet: 293T cells transfected with lentiviral vectors in the presence and absence of doxycycline (1 μg/ml) were collected 24 hours after transfection and analysed by Western blot as described by Abcam.

    Techniques: Concentration Assay, Transfection, Sequencing, Fluorescence

    hsa-miR-29a-3p inhibited luciferase reporter gene expression controlled by the 3′-UTRs of ALDH5A1 or SLC22A7 (A) Free energy analyses for the interactions between hsa-miR-29a-3p and the targeting sequences or site-mutants present the in ALDH5A1 3′-UTR, or SLC22A7 3′-UTR. Δ G , free energy; the underlined letter, the mutated base. (B) ALDH5A1-CU and ALDH5A1-Mut plasmids or (C) SLC22A7-CU and SLC22A7-Mut plasmids were transiently transfected into 293T and HepG2 cells, together with 50 nmol/L hsa-miR-29a-3p mimic or miRNA negative control. Cells were harvested 48 h after transfection. Three independent experiments, each in triplicate, were performed, and fold changes of luciferase activity were calculated by defining the activity of ALDH5A1-CU plasmid, or SLC22A7-CU, together with miRNA negative control as unity. ** P

    Journal: Biochemical pharmacology

    Article Title: Modulation of ALDH5A1 and SLC22A7 by microRNA hsa-miR-29a-3p in human liver cells

    doi: 10.1016/j.bcp.2015.09.020

    Figure Lengend Snippet: hsa-miR-29a-3p inhibited luciferase reporter gene expression controlled by the 3′-UTRs of ALDH5A1 or SLC22A7 (A) Free energy analyses for the interactions between hsa-miR-29a-3p and the targeting sequences or site-mutants present the in ALDH5A1 3′-UTR, or SLC22A7 3′-UTR. Δ G , free energy; the underlined letter, the mutated base. (B) ALDH5A1-CU and ALDH5A1-Mut plasmids or (C) SLC22A7-CU and SLC22A7-Mut plasmids were transiently transfected into 293T and HepG2 cells, together with 50 nmol/L hsa-miR-29a-3p mimic or miRNA negative control. Cells were harvested 48 h after transfection. Three independent experiments, each in triplicate, were performed, and fold changes of luciferase activity were calculated by defining the activity of ALDH5A1-CU plasmid, or SLC22A7-CU, together with miRNA negative control as unity. ** P

    Article Snippet: The 293T cells were obtained from Biosettia (San Diego, CA) and maintained in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% FBS, 1 mM sodium pyruvate, and non-essential amino acids.

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

    Quantitative analysis of TGF-β pathway activation by aqueous humor of patients and controls. Box and whisker plot showing that the aqueous humor of naïve nAMD patients induces less TGF-β pathway activation in Lenti-X 293T cells. Cells were co-transfected with the plasmids pNL[NlucP/SBE], expressing NanoLuc luciferase under the control of three SMAD3 binding elements, and pGL4.54[luc2/TK], expressing the Firefly luciferase luc2 (used as transfection normalizer) under the control of the constitutive HSV-TK promoter. Transfected cells were treated with 10 μl of aqueous humor of the 20 nAMD patients, naïve or treated (once or twice, Treat.1 and Treat.2, respectively), or of the 20 control samples (Cntr.) described in Table 1 . Data are presented as the ratio between NanoLuc and luc2 luciferase activity, expressed as Relative Light Units (RLU) (number of replicates for each sample = 3). Asterisks indicate significant differences (**P

    Journal: Scientific Reports

    Article Title: TGF-β concentrations and activity are down-regulated in the aqueous humor of patients with neovascular age-related macular degeneration

    doi: 10.1038/s41598-018-26442-0

    Figure Lengend Snippet: Quantitative analysis of TGF-β pathway activation by aqueous humor of patients and controls. Box and whisker plot showing that the aqueous humor of naïve nAMD patients induces less TGF-β pathway activation in Lenti-X 293T cells. Cells were co-transfected with the plasmids pNL[NlucP/SBE], expressing NanoLuc luciferase under the control of three SMAD3 binding elements, and pGL4.54[luc2/TK], expressing the Firefly luciferase luc2 (used as transfection normalizer) under the control of the constitutive HSV-TK promoter. Transfected cells were treated with 10 μl of aqueous humor of the 20 nAMD patients, naïve or treated (once or twice, Treat.1 and Treat.2, respectively), or of the 20 control samples (Cntr.) described in Table 1 . Data are presented as the ratio between NanoLuc and luc2 luciferase activity, expressed as Relative Light Units (RLU) (number of replicates for each sample = 3). Asterisks indicate significant differences (**P

    Article Snippet: Analysis of TGF-β pathway activation The luciferase-based reporter system used to analyze activation of the TGF-β pathway consisted of Lenti-X 293T cells (#632180; Takara Bio, CA) transiently transfected with the plasmid pNL[NlucP/SBE] (Promega Corp., Madison, WI), carrying three copies of the SMAD binding element (SBE) (AGTATGTCTAGACTGA) which represent specific targets of for phospho-SMAD3 and drive the expression of a reporter gene encoding NanoLuc® Luciferase.

    Techniques: Activation Assay, Whisker Assay, Transfection, Expressing, Luciferase, Binding Assay, Activity Assay

    TP15-Fc specifically binds human, but not mouse ICAM-1 (A) Flow cytometric analyses of non-transfected and stably transfected CHO-K1 cells with varying ICAM-1 expression levels detected by CD54-FITC (Beckman Coulter; dark grey) correlated with binding of 10 μg/ml TP15-Fc (light grey). 4D5-Fc was used as control (black). (B) ICAM-1 expression of non-transfected (upper row, left) and human ICAM-1 transfected Lenti-X 293T cells (upper row, middle) was measured by flow cytometry using mouse anti-human CD54-FITC (grey) and a FITC-labeled isotype control (black). Mouse ICAM-1 was expressed as GFP-tagged protein and positive cells were detected by fluorescence (upper row, right; grey histogram). Non-transfected cells served as control (black). Binding of 100 μg/ml TP15-Fc (grey) and 4D5-Fc (black), respectively, on non-transfected and ICAM-1-positive cell populations was detected with a PE-labeled anti-human Fcγ-specific secondary antibody (lower panel).

    Journal: Oncotarget

    Article Title: A novel Fc-engineered human ICAM-1/CD54 antibody with potent anti-myeloma activity developed by cellular panning of phage display libraries

    doi: 10.18632/oncotarget.20641

    Figure Lengend Snippet: TP15-Fc specifically binds human, but not mouse ICAM-1 (A) Flow cytometric analyses of non-transfected and stably transfected CHO-K1 cells with varying ICAM-1 expression levels detected by CD54-FITC (Beckman Coulter; dark grey) correlated with binding of 10 μg/ml TP15-Fc (light grey). 4D5-Fc was used as control (black). (B) ICAM-1 expression of non-transfected (upper row, left) and human ICAM-1 transfected Lenti-X 293T cells (upper row, middle) was measured by flow cytometry using mouse anti-human CD54-FITC (grey) and a FITC-labeled isotype control (black). Mouse ICAM-1 was expressed as GFP-tagged protein and positive cells were detected by fluorescence (upper row, right; grey histogram). Non-transfected cells served as control (black). Binding of 100 μg/ml TP15-Fc (grey) and 4D5-Fc (black), respectively, on non-transfected and ICAM-1-positive cell populations was detected with a PE-labeled anti-human Fcγ-specific secondary antibody (lower panel).

    Article Snippet: Lenti-X 293T (Clontech, Mountain View, CA, USA) and CHO-K1 cells were cultured in DMEM (Life Technologies) containing 10% FCS and antibiotics (D10+).

    Techniques: Flow Cytometry, Transfection, Stable Transfection, Expressing, Binding Assay, Cytometry, Labeling, Fluorescence

    DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) HEK-293 cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1

    Journal: Scientific Reports

    Article Title: Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2

    doi: 10.1038/srep43471

    Figure Lengend Snippet: DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) HEK-293 cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1

    Article Snippet: HEK 293 cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml).

    Techniques: In Vitro, In Vivo, Incubation, Western Blot, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Immunoprecipitation

    Coexpression of ERK3 or ERK4 results in cytoplasmatic relocalisation of DUSP2. ( a ) HEK293 cells were transfected with expression plasmids encoding myc-tagged catalytically inactive DUSP2 mutant (DUSP2CS), EGFP-tagged ERK3, ERK4 or a combination of DUSP2CS and ERK3 or DUSP2CS and ERK4. After 24 h the cells were fixed, EGFP fluorescence was visualized directly, and myc-tagged DUSP2 was visualized by staining with an anti-myc antibody (9E10) and Alexa-594 anti mouse antibody. ( b ). HEK293 cells were cotransfected with expression plasmid encoding either myc-tagged kinase interaction motif-deficient mutant of inactive DUSP2 (DUSP2CS-KIM) and EGFP-tagged ERK3 or myc-tagged kinase interaction motif-deficient mutant of inactive DUSP2(DUSP2CS-KIM) and EGFP-tagged ERK4. The cell was fixed and protein visualized as in A. ( c ) HEK 293 cells were co-transfected with the indicated expression vectors, and EGFP-ERK3, EGFP-ERK4, myc-DUSP2CS and myc-DUSP2CS-KIM were visualized as above. In all, more than 100 cells expressing DUSP2CS or DUSP2CS-KIM alone or together with either EGFP-ERK3 or EGFP-ERK4 from three independent transfections were counted, and the distribution of DUSP2 protein was scored in percentages. The results are presented as the percentages of cells in which myc-DUSP2 was predominantly cytosolic (C > N), and the mean values with the associated SD are shown. In all experiments, 10 different fields of cells were examined, and the representative images are as shown in Fig.6a and b.

    Journal: Scientific Reports

    Article Title: Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2

    doi: 10.1038/srep43471

    Figure Lengend Snippet: Coexpression of ERK3 or ERK4 results in cytoplasmatic relocalisation of DUSP2. ( a ) HEK293 cells were transfected with expression plasmids encoding myc-tagged catalytically inactive DUSP2 mutant (DUSP2CS), EGFP-tagged ERK3, ERK4 or a combination of DUSP2CS and ERK3 or DUSP2CS and ERK4. After 24 h the cells were fixed, EGFP fluorescence was visualized directly, and myc-tagged DUSP2 was visualized by staining with an anti-myc antibody (9E10) and Alexa-594 anti mouse antibody. ( b ). HEK293 cells were cotransfected with expression plasmid encoding either myc-tagged kinase interaction motif-deficient mutant of inactive DUSP2 (DUSP2CS-KIM) and EGFP-tagged ERK3 or myc-tagged kinase interaction motif-deficient mutant of inactive DUSP2(DUSP2CS-KIM) and EGFP-tagged ERK4. The cell was fixed and protein visualized as in A. ( c ) HEK 293 cells were co-transfected with the indicated expression vectors, and EGFP-ERK3, EGFP-ERK4, myc-DUSP2CS and myc-DUSP2CS-KIM were visualized as above. In all, more than 100 cells expressing DUSP2CS or DUSP2CS-KIM alone or together with either EGFP-ERK3 or EGFP-ERK4 from three independent transfections were counted, and the distribution of DUSP2 protein was scored in percentages. The results are presented as the percentages of cells in which myc-DUSP2 was predominantly cytosolic (C > N), and the mean values with the associated SD are shown. In all experiments, 10 different fields of cells were examined, and the representative images are as shown in Fig.6a and b.

    Article Snippet: HEK 293 cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml).

    Techniques: Transfection, Expressing, Mutagenesis, Fluorescence, Staining, Plasmid Preparation

    FOXO1 is a direct target of miR-582-5p. (A) Alignment between the predicted miR-582-5p target site of FOXO1 3′UTR region and miR-582-5p. (B) Real time RT-PCR and western blot analysis showing FOXO1 mRNA and protein expression levels in THP-1 cells transfected with miR-582-5p mimics or mimics NC, respectively. (C) Co-transfection of miR-582-5p mimics/mimics NC and FOXO1 3′UTR-luciferase reporter vector into HEK-293T cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence (FOXO1-3′UTR-wt), not in vector that contained a mutant sequence (FOXO1-3′UTR-mut) within the miR-582-5p binding site. Values were presented as relative luciferase activity after normalization to Renilla luciferase activity. FOXO1-3′UTR-wt: pMIR-FOXO1-3′UTR-wt vector; FOXO1-3′UTR-mut: pMIR-FOXO1-3′UTR-mut vector. (D) Representative flow cytometric plots showing apoptotic ratio of THP-1 transfected with FOXO1 siRNA or negative controls of siRNA (siRNA NC) (left panel). The apoptotic percentage of THP-1 cells transfected with FOXO1 siRNA were significantly lower than those transfected with negative control siRNA (siRNA NC) ( p

    Journal: PLoS ONE

    Article Title: miR-582-5p Is Upregulated in Patients with Active Tuberculosis and Inhibits Apoptosis of Monocytes by Targeting FOXO1

    doi: 10.1371/journal.pone.0078381

    Figure Lengend Snippet: FOXO1 is a direct target of miR-582-5p. (A) Alignment between the predicted miR-582-5p target site of FOXO1 3′UTR region and miR-582-5p. (B) Real time RT-PCR and western blot analysis showing FOXO1 mRNA and protein expression levels in THP-1 cells transfected with miR-582-5p mimics or mimics NC, respectively. (C) Co-transfection of miR-582-5p mimics/mimics NC and FOXO1 3′UTR-luciferase reporter vector into HEK-293T cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence (FOXO1-3′UTR-wt), not in vector that contained a mutant sequence (FOXO1-3′UTR-mut) within the miR-582-5p binding site. Values were presented as relative luciferase activity after normalization to Renilla luciferase activity. FOXO1-3′UTR-wt: pMIR-FOXO1-3′UTR-wt vector; FOXO1-3′UTR-mut: pMIR-FOXO1-3′UTR-mut vector. (D) Representative flow cytometric plots showing apoptotic ratio of THP-1 transfected with FOXO1 siRNA or negative controls of siRNA (siRNA NC) (left panel). The apoptotic percentage of THP-1 cells transfected with FOXO1 siRNA were significantly lower than those transfected with negative control siRNA (siRNA NC) ( p

    Article Snippet: Human monocytic cell line THP-1 (TIB-202) and human embryonic kidney 293T cells (CRL-11268) were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Cotransfection, Luciferase, Plasmid Preparation, Activity Assay, Sequencing, Mutagenesis, Binding Assay, Flow Cytometry, Negative Control

    Construction strategies and secretion analysis of DENV1-4 VLPs . A) Schematic representation of three strategies to construct DENV1-4 VLP expression plasmids, pD1-D4prME include the entire prM and E gene regions of DENV. JEV signal sequence (JESS) is introduced to the N-terminal of pJD1-D4prME, and pJD1-D4prMEΔ20%JEV. The corresponding JEV-E sequence was constructed in pJD1-D4prMEΔ20%JEV to replace the 20% C-terminal region of DENV-E; B) Western blot analysis of DENV1-4 VLPs secretion, Concentrated culture supernatants from 293T cells transformed with pD1-D4prME, pJD1-D4prME or pJD1-D4prMEΔ20%JEV were tested by rabbit polyclonal serum specific against dengue E protein. Bound antibodies were detected by a HRP-conjugated goat anti-mouse antibody. The band corresponding to envelope (E) is indicated with an arrow to the right of the blot.

    Journal: Virology Journal

    Article Title: Vaccination with dengue virus-like particles induces humoral and cellular immune responses in mice

    doi: 10.1186/1743-422X-8-333

    Figure Lengend Snippet: Construction strategies and secretion analysis of DENV1-4 VLPs . A) Schematic representation of three strategies to construct DENV1-4 VLP expression plasmids, pD1-D4prME include the entire prM and E gene regions of DENV. JEV signal sequence (JESS) is introduced to the N-terminal of pJD1-D4prME, and pJD1-D4prMEΔ20%JEV. The corresponding JEV-E sequence was constructed in pJD1-D4prMEΔ20%JEV to replace the 20% C-terminal region of DENV-E; B) Western blot analysis of DENV1-4 VLPs secretion, Concentrated culture supernatants from 293T cells transformed with pD1-D4prME, pJD1-D4prME or pJD1-D4prMEΔ20%JEV were tested by rabbit polyclonal serum specific against dengue E protein. Bound antibodies were detected by a HRP-conjugated goat anti-mouse antibody. The band corresponding to envelope (E) is indicated with an arrow to the right of the blot.

    Article Snippet: Cells and viruses 293T cells (ATCC No.CRL-11268) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C with 5% CO2.

    Techniques: Construct, Expressing, Sequencing, Western Blot, Transformation Assay

    Human Jurkat T cells lentivirally transduced with Runx1.d190 show increased transcription of c-Myc . (A) Schematic of the structure of Runx1 and Runx1.d190. (B) 293T. cells were transfected with empty pEGFP-N1 vector (EGFPonly, left column) as a control for cytoplasmic staining, pEGFP-N1 vector containing full-length Runx1 fused in-frame to EGFP (Runx1FL, middle column) or Runx1.d190 fused in-frame to EGFP (Runx1.d190, right column). The nuclear DNA was visualized by staining with Hoescht 33342 (Nuclear, top row). Nuclear (top row) and EGFP (middle row) fluorescence are shown in isolation and merged (Merged, bottom row). (C) Relative differences in transcription between Jurkat T cells lentivirally transduced with control empty vector or vector encoding Runx1.d190 as determined by microarray analysis are shown. A complete listing of genes whose transcription is affected by Runx1.d190 in Jurkat T cells is located at http://www.ncbi.nlm.nih.gov/geo/ . (D) ChIP analysis. Chromatin was prepared from Jurkat T cells lentivirally transduced with Runx1.d190 and immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out using primer sets amplifying Runx1-binding sites at -0.83 (i), -7.9 (ii) and -8.9 kb (iii) upstream of the human c-Myc transcriptional start site. N =3.

    Journal: PLoS ONE

    Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

    doi: 10.1371/journal.pone.0069083

    Figure Lengend Snippet: Human Jurkat T cells lentivirally transduced with Runx1.d190 show increased transcription of c-Myc . (A) Schematic of the structure of Runx1 and Runx1.d190. (B) 293T. cells were transfected with empty pEGFP-N1 vector (EGFPonly, left column) as a control for cytoplasmic staining, pEGFP-N1 vector containing full-length Runx1 fused in-frame to EGFP (Runx1FL, middle column) or Runx1.d190 fused in-frame to EGFP (Runx1.d190, right column). The nuclear DNA was visualized by staining with Hoescht 33342 (Nuclear, top row). Nuclear (top row) and EGFP (middle row) fluorescence are shown in isolation and merged (Merged, bottom row). (C) Relative differences in transcription between Jurkat T cells lentivirally transduced with control empty vector or vector encoding Runx1.d190 as determined by microarray analysis are shown. A complete listing of genes whose transcription is affected by Runx1.d190 in Jurkat T cells is located at http://www.ncbi.nlm.nih.gov/geo/ . (D) ChIP analysis. Chromatin was prepared from Jurkat T cells lentivirally transduced with Runx1.d190 and immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out using primer sets amplifying Runx1-binding sites at -0.83 (i), -7.9 (ii) and -8.9 kb (iii) upstream of the human c-Myc transcriptional start site. N =3.

    Article Snippet: Transfection and staining of 293T cells 293T cells were transfected with 6 µL of FuGene HD (Roche, Indianapolis, IN) and 1 µg of pEGFP-N1 vector alone, or pEGFP-N1 with Runx1.d190 or full-length Runx1 fused in frame to the 5’ end of EGFP as per manufacturer’s instructions.

    Techniques: Transduction, Transfection, Plasmid Preparation, Staining, Fluorescence, Isolation, Microarray, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Binding Assay