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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 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    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: 96/100, based on 37214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 3248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 9499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam 293t cells
    Characterization of the PIAS1 antibody. (A) PIAS1 and actin-immunoblots of lysate of <t>293T</t> cells expressing one of two different concentrations (1X or 10X) of FLAG-tagged PIAS1, PIAS2-xα, PIAS2-xβ, PIAS3 or, PIAS4, or transfected with the vector control. Actin immunoblotting was used as loading control. Only PIAS1 antibody-immunoreactive bands corresponding to endogenous PIAS1 and exogenous FLAG tagged-PIAS1 were detected. Immunoblots are from an experiment that was repeated twice with similar results. (B) PIAS1 or actin immunoblots of lysate of MDA-MB-231 cells transiently transfected with an RNAi vector control or receiving a pool of plasmids expressing short hairpin RNAs against two distinct regions of PIAS1 [ 18 , 19 ]. Control and PIAS1 RNAi plasmids also express CMV-driven green fluorescence protein (GFP). Immunoblots are from an experiment that was repeated twice with similar results. (C) Representative PIAS1 (red), GFP (green) and nuclei (blue) fluorescence microscopy micrographs of MDA-MB-231 cells transfected as in B, and subjected to anti-PIAS1 indirect immunofluorescence and counterstained with Hoechst 33342 fluorescent nucleotide dye to visualize nuclei. GFP signal indicate control vector or PIAS1 RNAi plasmid-transfected cells. Arrow shows an example of each a vector transfected cell (upper row) and a PIAS1 RNAi transfected cell (lower row) to highlight the knockdown of endogenous PIAS1 in PIAS1i-transfected cell. These experiments were repeated two times with similar outcomes. (D) Representative PIAS1 immunoblots of serially-diluted lysate of 293T cells (upper panel), and protein abundance of PIAS1 quantified by densitometry (y-axis) plotted versus the protein amount of lysate (x-axis) (lower panel). The abundance of PIAS1 for each point in the XY graph is the mean ± SEM from three independent experiments including the one shown in upper panel. Regression analysis indicated that the protein abundance of PIAS1 follows a linear relationship with total protein amount in cells lysates.
    293t Cells, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen 293t cells
    Anti-Celiac Peptide Antibodies Activate TLR4 in Cells Transfected with the TLR4 Gene Activation of NF-κB upon engagement of TLR4. (A) Stimulation of <t>293T</t> cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4); by affinity-purified anti-celiac peptide antibodies, 4 μg/ml (5), 2 μg/ml (6), 1 μg/ml (7), 0.5 μg/ml (8) by affinity-purified antibodies directed against an irrelevant control peptide, 4 μg/ml (9), 2 μg/ml (10), 1 μg/ml (11), and 0.5 μg/ml (12). (B) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4), by affinity-purified antibodies against an irrelevant peptide 1 μg/ml (5), by affinity-purified anti-celiac peptide antibodies 1 μg/ml (6), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml anti-TLR4 monoclonal antibody (7), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml celiac peptide (8), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml irrelevant control peptide (9), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml recombinant human tTG (10), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml (11), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml in the presence of 1 μg/ml VP-7 peptide (12), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml in the presence of 1 μg/ml celiac peptide (13), and by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml ovalbumin (14). Results are expressed as percentage of positive control, where the positive control is the OD value obtained upon stimulation of TLR4 transfected cells with 100 ng/ml LPS (maximal concentration used).
    293t Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson 293t cells
    Vpu-mediated down-regulation of CD1d correlates with decreased activation of CD1d-restricted iNKT cells. <t>293T</t> cells were co-transfected with CD1d and HIV-1 vpu alleles or eGFP control, respectively. At 24 h post transfection, cells were used for analysis of CD1d surface expression levels using flow cytometry and loading with the model lipid antigen αGalCer. The human CD4 + iNKT cell clone HDD3 was co-incubated with the αGalCer-loaded 293T cells in the presence of brefeldin A for 6 h, and subsequently analyzed for IFN-γ production using flow cytometry. iNKT cell IFN-γ production induced by 293T cells co-transfected with CD1d and eGFP control was set to 1, and all samples were calculated relative to control. (a) Representative dot plots showing iNKT cell IFN-γ production induced by 293T cells co-transfected with CD1d and vpu alleles of the indicated HIV-1 groups and controls. (b) Relationship between Vpu-mediated CD1d down-regulation and iNKT cell activation was assessed using linear regression and Pearson correlation. (c) Effect of expression of different HIV-1 vpu alleles on IFN-γ production by iNKT cells. Blue and green symbols represent subtype B and C Vpu proteins, respectively. Each data point represents one vpu allele and the average value of two experiments performed in at least duplicate.
    293t Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech 293t cells
    Vpu-mediated down-regulation of CD1d correlates with decreased activation of CD1d-restricted iNKT cells. <t>293T</t> cells were co-transfected with CD1d and HIV-1 vpu alleles or eGFP control, respectively. At 24 h post transfection, cells were used for analysis of CD1d surface expression levels using flow cytometry and loading with the model lipid antigen αGalCer. The human CD4 + iNKT cell clone HDD3 was co-incubated with the αGalCer-loaded 293T cells in the presence of brefeldin A for 6 h, and subsequently analyzed for IFN-γ production using flow cytometry. iNKT cell IFN-γ production induced by 293T cells co-transfected with CD1d and eGFP control was set to 1, and all samples were calculated relative to control. (a) Representative dot plots showing iNKT cell IFN-γ production induced by 293T cells co-transfected with CD1d and vpu alleles of the indicated HIV-1 groups and controls. (b) Relationship between Vpu-mediated CD1d down-regulation and iNKT cell activation was assessed using linear regression and Pearson correlation. (c) Effect of expression of different HIV-1 vpu alleles on IFN-γ production by iNKT cells. Blue and green symbols represent subtype B and C Vpu proteins, respectively. Each data point represents one vpu allele and the average value of two experiments performed in at least duplicate.
    293t Cells, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 92/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 293t cells
    Phosphatidylinositol-3-kinase (PI3K) and Akt are downstream of interferon (IFN) promoter stimulator-1 (IPS-1) activating IFN regulatory factor 3 (IRF3) activation and IFN- β expression. (a–d) <t>293T</t> cells were transfected with an expression
    293t Cells, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa aavpro 293t cells
    Phosphatidylinositol-3-kinase (PI3K) and Akt are downstream of interferon (IFN) promoter stimulator-1 (IPS-1) activating IFN regulatory factor 3 (IRF3) activation and IFN- β expression. (a–d) <t>293T</t> cells were transfected with an expression
    Aavpro 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza 293t cells
    PNDA-3 inhibits periostin-dependent signaling. ( a ) Schematic representation of the full-length periostin and deletion constructs used in this study. ( b ) Biotinylated PNDA-3 was mixed with cell lysates from <t>293T</t> cells overexpressing mutant periostins,
    293t Cells, supplied by Lonza, used in various techniques. Bioz Stars score: 93/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DSMZ 293t cells
    CDK9 controls Ser2P and is essential for activator-driven transcription. (A) ChIP profile of CTD Ser5P, and (B) Ser2P across the p21 and GAPDH loci in the absence or presence of 067. Lower graphs show relative levels of CTD modifications after normalizing to RNAPII. (C) ChIP profile of CDK9 (and, for comparison, of Ser2P). (D) Immunoblot (IB) analysis of RNAPII and bulk levels of Ser2P/5P/7P in control, CDK9 and CDK12 knockdown cells. Band intensities of CTD modifications were determined using ImageJ and normalized to RNAPII in the control sample. (E) Effect of CDK9 or CDK12 knockdown on p21 inducibility was determined as in Fig 3A . (F) Nuclear extracts (NE) from <t>293T</t> cells were immunodepleted (□) with non-specific IgG or the indicated antibodies and analyzed by immunoblotting (IB). (G) Depleted extracts were used in in vitro transcription reactions together with recombinant Gal4-VP16 activator and a responsive PCR template containing the Adenovirus major late promoter (AdML). Transcription elongation continued for 5 or 12 minutes, and radioactively labeled transcripts (tx) were visualized by autoradiography.
    293t Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 92/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cyagen Biosciences 293t cells
    CDK9 controls Ser2P and is essential for activator-driven transcription. (A) ChIP profile of CTD Ser5P, and (B) Ser2P across the p21 and GAPDH loci in the absence or presence of 067. Lower graphs show relative levels of CTD modifications after normalizing to RNAPII. (C) ChIP profile of CDK9 (and, for comparison, of Ser2P). (D) Immunoblot (IB) analysis of RNAPII and bulk levels of Ser2P/5P/7P in control, CDK9 and CDK12 knockdown cells. Band intensities of CTD modifications were determined using ImageJ and normalized to RNAPII in the control sample. (E) Effect of CDK9 or CDK12 knockdown on p21 inducibility was determined as in Fig 3A . (F) Nuclear extracts (NE) from <t>293T</t> cells were immunodepleted (□) with non-specific IgG or the indicated antibodies and analyzed by immunoblotting (IB). (G) Depleted extracts were used in in vitro transcription reactions together with recombinant Gal4-VP16 activator and a responsive PCR template containing the Adenovirus major late promoter (AdML). Transcription elongation continued for 5 or 12 minutes, and radioactively labeled transcripts (tx) were visualized by autoradiography.
    293t Cells, supplied by Cyagen Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hek 293 cells
    Membrane localization of EGFP-tagged Ca V 1.2e1b and Ca V 1.2e1c channels when expressed with α 2 δ or with α 2 δ + β 1b subunits in <t>HEK</t> 293 cells
    Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 6284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genechem 293t cells
    Membrane localization of EGFP-tagged Ca V 1.2e1b and Ca V 1.2e1c channels when expressed with α 2 δ or with α 2 δ + β 1b subunits in <t>HEK</t> 293 cells
    293t Cells, supplied by Genechem, used in various techniques. Bioz Stars score: 92/100, based on 373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    System Biosciences Inc 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
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    GenePharma Company 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
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    Orbigen 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 Orbigen, used in various techniques. Bioz Stars score: 92/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    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

    Roc-A inhibits the activity of the Rho GTPases RhoA, Rac1 and Cdc42 A. Roc-A inhibits the activity of the Rho GTPases RhoA, Rac1 and Cdc42. RhoA, Rac1 or Cdc42 FRET sensors were overexpressed in 293T cells either with a control plasmid (Control, 30 nM Roc-A) or together with GDI. Cells were treated with 30 nM Roc-A or vehicle (DMSO for Control and GDI) for 24h. Representative fluorescence micrographs show mVenus channel of 293T cells expressing the indicated FRET sensors. Scale bars: 100 μm. Pseudocolored FRET efficiency images of the same field of view were calculated as a ratio of FRET acceptor over FRET donor emission intensity reflecting the GTPase activity levels. The histograms show the pixel distribution of the FRET efficiency within the FRET emission ratio images. B. Quantification of A. FRET efficiency was normalized to GDI and DMSO values and % inhibition FRET efficiency was calculated. Results are an average of six independent experiments. Error bars (S.E.M.) are shown. C. Roc-A inhibits the activity of the Rho GTPases RhoA, Rac1 and Cdc42 in HeLa cells. RhoA, Rac1 or Cdc42 FRET sensors were overexpressed in HeLa cells either with a control plasmid (Control, 30 nM Roc-A) or together with GDI. Cells were treated with 30 nM Roc-A or vehicle (DMSO for Control and GDI) for 24h. FRET efficiency was normalized to GDI and DMSO values and % inhibition FRET efficiency was calculated. Results are an average of three independent experiments. Error bars (S.E.M.) are shown.

    Journal: Oncotarget

    Article Title: The anticancer phytochemical rocaglamide inhibits Rho GTPase activity and cancer cell migration

    doi: 10.18632/oncotarget.10188

    Figure Lengend Snippet: Roc-A inhibits the activity of the Rho GTPases RhoA, Rac1 and Cdc42 A. Roc-A inhibits the activity of the Rho GTPases RhoA, Rac1 and Cdc42. RhoA, Rac1 or Cdc42 FRET sensors were overexpressed in 293T cells either with a control plasmid (Control, 30 nM Roc-A) or together with GDI. Cells were treated with 30 nM Roc-A or vehicle (DMSO for Control and GDI) for 24h. Representative fluorescence micrographs show mVenus channel of 293T cells expressing the indicated FRET sensors. Scale bars: 100 μm. Pseudocolored FRET efficiency images of the same field of view were calculated as a ratio of FRET acceptor over FRET donor emission intensity reflecting the GTPase activity levels. The histograms show the pixel distribution of the FRET efficiency within the FRET emission ratio images. B. Quantification of A. FRET efficiency was normalized to GDI and DMSO values and % inhibition FRET efficiency was calculated. Results are an average of six independent experiments. Error bars (S.E.M.) are shown. C. Roc-A inhibits the activity of the Rho GTPases RhoA, Rac1 and Cdc42 in HeLa cells. RhoA, Rac1 or Cdc42 FRET sensors were overexpressed in HeLa cells either with a control plasmid (Control, 30 nM Roc-A) or together with GDI. Cells were treated with 30 nM Roc-A or vehicle (DMSO for Control and GDI) for 24h. FRET efficiency was normalized to GDI and DMSO values and % inhibition FRET efficiency was calculated. Results are an average of three independent experiments. Error bars (S.E.M.) are shown.

    Article Snippet: Cell culture and reagents The non-tumor fibroblast cell line NIH-3T3, the human cell lines PC-3 (prostate cancer), MDA-MB-231 (breast cancer), the human colon cancer cell line HCT116, the human cervical cancer cell line HeLa, and 293T (transformed embryonic kidney) were cultured at 37°C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, Munich, Germany) supplemented with 10 % FCS (PC-3 and MDA-MB-231) or in DMEM medium (Sigma-Aldrich) supplemented with 10 % FCS (293T) until otherwise stated in the text.

    Techniques: Activity Assay, Plasmid Preparation, Fluorescence, Expressing, Inhibition

    Roc-A inhibits migration of different types of cancer cells A and B. Roc-A inhibits cell migration in 293T and MDA-MB-231 cells. 293T and MDA-MB-231 cells were subjected to treatment with different drugs and the percentage of gap closure was quantified as described in Figure 1C and 1D . Representative images are shown. C. Analysis of the effects of different drugs on proliferation in 293T and MDM-MB-231 cells. The percentage of proliferative cells was determined and normalized to DMSO treatment. Results are an average of three independent experiments. Error bars (S.D.) are shown. D. Analysis of the effects of different drugs on cell viability. The percentage of viable cells was determined as AnxV − /7aad − cells 20 h (293T) and 24 h (MDA-MB-231) after drug treatment. Results are an average of three independent experiments. Error bars (S.D.) are shown.

    Journal: Oncotarget

    Article Title: The anticancer phytochemical rocaglamide inhibits Rho GTPase activity and cancer cell migration

    doi: 10.18632/oncotarget.10188

    Figure Lengend Snippet: Roc-A inhibits migration of different types of cancer cells A and B. Roc-A inhibits cell migration in 293T and MDA-MB-231 cells. 293T and MDA-MB-231 cells were subjected to treatment with different drugs and the percentage of gap closure was quantified as described in Figure 1C and 1D . Representative images are shown. C. Analysis of the effects of different drugs on proliferation in 293T and MDM-MB-231 cells. The percentage of proliferative cells was determined and normalized to DMSO treatment. Results are an average of three independent experiments. Error bars (S.D.) are shown. D. Analysis of the effects of different drugs on cell viability. The percentage of viable cells was determined as AnxV − /7aad − cells 20 h (293T) and 24 h (MDA-MB-231) after drug treatment. Results are an average of three independent experiments. Error bars (S.D.) are shown.

    Article Snippet: Cell culture and reagents The non-tumor fibroblast cell line NIH-3T3, the human cell lines PC-3 (prostate cancer), MDA-MB-231 (breast cancer), the human colon cancer cell line HCT116, the human cervical cancer cell line HeLa, and 293T (transformed embryonic kidney) were cultured at 37°C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, Munich, Germany) supplemented with 10 % FCS (PC-3 and MDA-MB-231) or in DMEM medium (Sigma-Aldrich) supplemented with 10 % FCS (293T) until otherwise stated in the text.

    Techniques: Migration, Multiple Displacement Amplification

    Roc-A alters the morphology of F-actin-based protrusions by an indirect effect on actin polymerization A. Roc-A alters the morphology of F-actin-based protrusions. PC-3, MDA-MB-231 and 293T cells were treated with Roc-A (15 nM for PC3, 60 nM for MDA-MB-231 and 30 nM for 293T) or solvent (DMSO) for 24h followed by staining of F-actin (green) and nuclei (blue). Three independent experiments were carried out per cell line. Representative images (Z-stacks) per cell line are shown. Scale bar = 20 μm (PC-3) or 10 μm (293T and MDA-MB-231). B. Roc-A does not directly affect actin polymerization. The influence of Roc-A on polymerization of pyrene-conjugated actin monomers was monitored by measuring the increase in fluorescence that occurs upon polymerization of pyrene-conjugated actin. Roc-A, solvent control or the known actin polymerization promoting agent Jasplakinolide were added to pyrene-conjugated actin monomers and 20 min later actin polymerization was initiated. Results are representative of three independent experiments. C. Roc-A does not directly affect actin depolymerization. The influence of Roc-A on depolymerization of pyrene-conjugated actin monomers was monitored by measuring the decrease in fluorescence that occurs upon depolymeization of pyrene-conjugated actin. Roc-A, solvent control or the known F-actin stabilizer phalloidin were added to polymerized pyrene-conjugated actin for 20 min, after which depolymerization of actin was initiated. Results are representative of three independent experiments.

    Journal: Oncotarget

    Article Title: The anticancer phytochemical rocaglamide inhibits Rho GTPase activity and cancer cell migration

    doi: 10.18632/oncotarget.10188

    Figure Lengend Snippet: Roc-A alters the morphology of F-actin-based protrusions by an indirect effect on actin polymerization A. Roc-A alters the morphology of F-actin-based protrusions. PC-3, MDA-MB-231 and 293T cells were treated with Roc-A (15 nM for PC3, 60 nM for MDA-MB-231 and 30 nM for 293T) or solvent (DMSO) for 24h followed by staining of F-actin (green) and nuclei (blue). Three independent experiments were carried out per cell line. Representative images (Z-stacks) per cell line are shown. Scale bar = 20 μm (PC-3) or 10 μm (293T and MDA-MB-231). B. Roc-A does not directly affect actin polymerization. The influence of Roc-A on polymerization of pyrene-conjugated actin monomers was monitored by measuring the increase in fluorescence that occurs upon polymerization of pyrene-conjugated actin. Roc-A, solvent control or the known actin polymerization promoting agent Jasplakinolide were added to pyrene-conjugated actin monomers and 20 min later actin polymerization was initiated. Results are representative of three independent experiments. C. Roc-A does not directly affect actin depolymerization. The influence of Roc-A on depolymerization of pyrene-conjugated actin monomers was monitored by measuring the decrease in fluorescence that occurs upon depolymeization of pyrene-conjugated actin. Roc-A, solvent control or the known F-actin stabilizer phalloidin were added to polymerized pyrene-conjugated actin for 20 min, after which depolymerization of actin was initiated. Results are representative of three independent experiments.

    Article Snippet: Cell culture and reagents The non-tumor fibroblast cell line NIH-3T3, the human cell lines PC-3 (prostate cancer), MDA-MB-231 (breast cancer), the human colon cancer cell line HCT116, the human cervical cancer cell line HeLa, and 293T (transformed embryonic kidney) were cultured at 37°C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, Munich, Germany) supplemented with 10 % FCS (PC-3 and MDA-MB-231) or in DMEM medium (Sigma-Aldrich) supplemented with 10 % FCS (293T) until otherwise stated in the text.

    Techniques: Multiple Displacement Amplification, Staining, Fluorescence

    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

    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

    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

    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

    Characterization of the PIAS1 antibody. (A) PIAS1 and actin-immunoblots of lysate of 293T cells expressing one of two different concentrations (1X or 10X) of FLAG-tagged PIAS1, PIAS2-xα, PIAS2-xβ, PIAS3 or, PIAS4, or transfected with the vector control. Actin immunoblotting was used as loading control. Only PIAS1 antibody-immunoreactive bands corresponding to endogenous PIAS1 and exogenous FLAG tagged-PIAS1 were detected. Immunoblots are from an experiment that was repeated twice with similar results. (B) PIAS1 or actin immunoblots of lysate of MDA-MB-231 cells transiently transfected with an RNAi vector control or receiving a pool of plasmids expressing short hairpin RNAs against two distinct regions of PIAS1 [ 18 , 19 ]. Control and PIAS1 RNAi plasmids also express CMV-driven green fluorescence protein (GFP). Immunoblots are from an experiment that was repeated twice with similar results. (C) Representative PIAS1 (red), GFP (green) and nuclei (blue) fluorescence microscopy micrographs of MDA-MB-231 cells transfected as in B, and subjected to anti-PIAS1 indirect immunofluorescence and counterstained with Hoechst 33342 fluorescent nucleotide dye to visualize nuclei. GFP signal indicate control vector or PIAS1 RNAi plasmid-transfected cells. Arrow shows an example of each a vector transfected cell (upper row) and a PIAS1 RNAi transfected cell (lower row) to highlight the knockdown of endogenous PIAS1 in PIAS1i-transfected cell. These experiments were repeated two times with similar outcomes. (D) Representative PIAS1 immunoblots of serially-diluted lysate of 293T cells (upper panel), and protein abundance of PIAS1 quantified by densitometry (y-axis) plotted versus the protein amount of lysate (x-axis) (lower panel). The abundance of PIAS1 for each point in the XY graph is the mean ± SEM from three independent experiments including the one shown in upper panel. Regression analysis indicated that the protein abundance of PIAS1 follows a linear relationship with total protein amount in cells lysates.

    Journal: PLoS ONE

    Article Title: Identification of the SUMO E3 ligase PIAS1 as a potential survival biomarker in breast cancer

    doi: 10.1371/journal.pone.0177639

    Figure Lengend Snippet: Characterization of the PIAS1 antibody. (A) PIAS1 and actin-immunoblots of lysate of 293T cells expressing one of two different concentrations (1X or 10X) of FLAG-tagged PIAS1, PIAS2-xα, PIAS2-xβ, PIAS3 or, PIAS4, or transfected with the vector control. Actin immunoblotting was used as loading control. Only PIAS1 antibody-immunoreactive bands corresponding to endogenous PIAS1 and exogenous FLAG tagged-PIAS1 were detected. Immunoblots are from an experiment that was repeated twice with similar results. (B) PIAS1 or actin immunoblots of lysate of MDA-MB-231 cells transiently transfected with an RNAi vector control or receiving a pool of plasmids expressing short hairpin RNAs against two distinct regions of PIAS1 [ 18 , 19 ]. Control and PIAS1 RNAi plasmids also express CMV-driven green fluorescence protein (GFP). Immunoblots are from an experiment that was repeated twice with similar results. (C) Representative PIAS1 (red), GFP (green) and nuclei (blue) fluorescence microscopy micrographs of MDA-MB-231 cells transfected as in B, and subjected to anti-PIAS1 indirect immunofluorescence and counterstained with Hoechst 33342 fluorescent nucleotide dye to visualize nuclei. GFP signal indicate control vector or PIAS1 RNAi plasmid-transfected cells. Arrow shows an example of each a vector transfected cell (upper row) and a PIAS1 RNAi transfected cell (lower row) to highlight the knockdown of endogenous PIAS1 in PIAS1i-transfected cell. These experiments were repeated two times with similar outcomes. (D) Representative PIAS1 immunoblots of serially-diluted lysate of 293T cells (upper panel), and protein abundance of PIAS1 quantified by densitometry (y-axis) plotted versus the protein amount of lysate (x-axis) (lower panel). The abundance of PIAS1 for each point in the XY graph is the mean ± SEM from three independent experiments including the one shown in upper panel. Regression analysis indicated that the protein abundance of PIAS1 follows a linear relationship with total protein amount in cells lysates.

    Article Snippet: Immunoblotting analyses of 293T cells, in which increasing amount of lysate was analyzed, confirmed the utility of the Abcam antibody in recognizing endogenous PIAS1 in a linear dynamic range ( ).

    Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Multiple Displacement Amplification, Fluorescence, Microscopy, Immunofluorescence

    Anti-Celiac Peptide Antibodies Activate TLR4 in Cells Transfected with the TLR4 Gene Activation of NF-κB upon engagement of TLR4. (A) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4); by affinity-purified anti-celiac peptide antibodies, 4 μg/ml (5), 2 μg/ml (6), 1 μg/ml (7), 0.5 μg/ml (8) by affinity-purified antibodies directed against an irrelevant control peptide, 4 μg/ml (9), 2 μg/ml (10), 1 μg/ml (11), and 0.5 μg/ml (12). (B) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4), by affinity-purified antibodies against an irrelevant peptide 1 μg/ml (5), by affinity-purified anti-celiac peptide antibodies 1 μg/ml (6), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml anti-TLR4 monoclonal antibody (7), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml celiac peptide (8), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml irrelevant control peptide (9), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml recombinant human tTG (10), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml (11), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml in the presence of 1 μg/ml VP-7 peptide (12), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml in the presence of 1 μg/ml celiac peptide (13), and by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml ovalbumin (14). Results are expressed as percentage of positive control, where the positive control is the OD value obtained upon stimulation of TLR4 transfected cells with 100 ng/ml LPS (maximal concentration used).

    Journal: PLoS Medicine

    Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

    doi: 10.1371/journal.pmed.0030358

    Figure Lengend Snippet: Anti-Celiac Peptide Antibodies Activate TLR4 in Cells Transfected with the TLR4 Gene Activation of NF-κB upon engagement of TLR4. (A) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4); by affinity-purified anti-celiac peptide antibodies, 4 μg/ml (5), 2 μg/ml (6), 1 μg/ml (7), 0.5 μg/ml (8) by affinity-purified antibodies directed against an irrelevant control peptide, 4 μg/ml (9), 2 μg/ml (10), 1 μg/ml (11), and 0.5 μg/ml (12). (B) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4), by affinity-purified antibodies against an irrelevant peptide 1 μg/ml (5), by affinity-purified anti-celiac peptide antibodies 1 μg/ml (6), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml anti-TLR4 monoclonal antibody (7), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml celiac peptide (8), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml irrelevant control peptide (9), by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml recombinant human tTG (10), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml (11), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml in the presence of 1 μg/ml VP-7 peptide (12), by affinity-purified anti-VP-7 peptide antibodies 1 μg/ml in the presence of 1 μg/ml celiac peptide (13), and by affinity-purified anti-celiac peptide antibodies 1 μg/ml in the presence of 1 μg/ml ovalbumin (14). Results are expressed as percentage of positive control, where the positive control is the OD value obtained upon stimulation of TLR4 transfected cells with 100 ng/ml LPS (maximal concentration used).

    Article Snippet: Anti-Celiac Peptide Antibodies Activate TLR4 in Cells Stably Transfected with the TLR4 Gene 293T cells stably transfected with the genes coding for TLR4 and the co-receptors MD2 and CD14 (Invivogen) as well as 293T cells expressing the TLR9 gene were co-transfected with the pNifty plasmid (Invivogen) and stable transfectants selected with Zeocin.

    Techniques: Transfection, Activation Assay, Affinity Purification, Recombinant, Positive Control, Concentration Assay

    Antibodies against the Celiac Peptide Bind the Self-Antigens MPMR2 and TLR4 (A) Sera of patients with active CD contain IgA antibodies directed against MPMR2; such reactivity is not present in patients on GFD. A K562 cell lysate was probed with rabbit antiserum raised against a peptide (VEKIGGASSRGE) of the MPMR2 (Lane 1), with antibodies affinity-purified against the celiac peptide (lane 2), with antibodies affinity-purified against an irrelevant control peptide (Lane 3), with sera from patients with active disease on GCD (Lanes 4, 6, and 8), and with sera from the same patients on GFD (Lanes 5, 7, and 9). A peroxidase-labelled polyvalent anti-human Igs antibody (Lanes 2 and 3) and an anti-human IgA antibody (Lanes 4–9) were used for detection. (B) Cell lysate from untransfected 293T cells was probed with the monoclonal antibody against TLR4 (Lane 1). Cell lysate from 293T cells transfected with the human TLR4 gene was probed with a monoclonal antibody directed against TLR4 (Lane 2), and with antibodies affinity-purified against the celiac peptide (Lane 3). Cell lysate from 293T cells transfected with the TLR4 gene was probed with biotin-labelled anti-TLR4 monoclonal antibody (Lane 4). Cell lysate from 293T cells transfected with the TLR4 gene was probed first with affinity-purified anti-peptide antibodies, followed by an incubation with biotin-labelled anti-TLR4 monoclonal antibody (Lane 5). Cell lysate from human plasmocytoid dendritic cells was probed with the monoclonal antibody against TLR4 (Lane 6). Cell lysate from human monocytes was probed with the monoclonal antibody against TLR4 (Lane 7) and with affinity-purified anti-peptide antibodies (Lane 8). (C) Inhibition of binding of anti-celiac peptide antibodies to solid-phase TLR4 peptide by liquid-phase TLR4 peptide (blue line), by celiac peptide (red line), and VP-7 peptide (red line), but not by an irrelevant control peptide (green line). The y -axis represents percentage of inhibition, and the x -axis indicates inhibitor concentration (μg/ml).

    Journal: PLoS Medicine

    Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

    doi: 10.1371/journal.pmed.0030358

    Figure Lengend Snippet: Antibodies against the Celiac Peptide Bind the Self-Antigens MPMR2 and TLR4 (A) Sera of patients with active CD contain IgA antibodies directed against MPMR2; such reactivity is not present in patients on GFD. A K562 cell lysate was probed with rabbit antiserum raised against a peptide (VEKIGGASSRGE) of the MPMR2 (Lane 1), with antibodies affinity-purified against the celiac peptide (lane 2), with antibodies affinity-purified against an irrelevant control peptide (Lane 3), with sera from patients with active disease on GCD (Lanes 4, 6, and 8), and with sera from the same patients on GFD (Lanes 5, 7, and 9). A peroxidase-labelled polyvalent anti-human Igs antibody (Lanes 2 and 3) and an anti-human IgA antibody (Lanes 4–9) were used for detection. (B) Cell lysate from untransfected 293T cells was probed with the monoclonal antibody against TLR4 (Lane 1). Cell lysate from 293T cells transfected with the human TLR4 gene was probed with a monoclonal antibody directed against TLR4 (Lane 2), and with antibodies affinity-purified against the celiac peptide (Lane 3). Cell lysate from 293T cells transfected with the TLR4 gene was probed with biotin-labelled anti-TLR4 monoclonal antibody (Lane 4). Cell lysate from 293T cells transfected with the TLR4 gene was probed first with affinity-purified anti-peptide antibodies, followed by an incubation with biotin-labelled anti-TLR4 monoclonal antibody (Lane 5). Cell lysate from human plasmocytoid dendritic cells was probed with the monoclonal antibody against TLR4 (Lane 6). Cell lysate from human monocytes was probed with the monoclonal antibody against TLR4 (Lane 7) and with affinity-purified anti-peptide antibodies (Lane 8). (C) Inhibition of binding of anti-celiac peptide antibodies to solid-phase TLR4 peptide by liquid-phase TLR4 peptide (blue line), by celiac peptide (red line), and VP-7 peptide (red line), but not by an irrelevant control peptide (green line). The y -axis represents percentage of inhibition, and the x -axis indicates inhibitor concentration (μg/ml).

    Article Snippet: Anti-Celiac Peptide Antibodies Activate TLR4 in Cells Stably Transfected with the TLR4 Gene 293T cells stably transfected with the genes coding for TLR4 and the co-receptors MD2 and CD14 (Invivogen) as well as 293T cells expressing the TLR9 gene were co-transfected with the pNifty plasmid (Invivogen) and stable transfectants selected with Zeocin.

    Techniques: Affinity Purification, Transfection, Incubation, Inhibition, Binding Assay, Concentration Assay

    Vpu-mediated down-regulation of CD1d correlates with decreased activation of CD1d-restricted iNKT cells. 293T cells were co-transfected with CD1d and HIV-1 vpu alleles or eGFP control, respectively. At 24 h post transfection, cells were used for analysis of CD1d surface expression levels using flow cytometry and loading with the model lipid antigen αGalCer. The human CD4 + iNKT cell clone HDD3 was co-incubated with the αGalCer-loaded 293T cells in the presence of brefeldin A for 6 h, and subsequently analyzed for IFN-γ production using flow cytometry. iNKT cell IFN-γ production induced by 293T cells co-transfected with CD1d and eGFP control was set to 1, and all samples were calculated relative to control. (a) Representative dot plots showing iNKT cell IFN-γ production induced by 293T cells co-transfected with CD1d and vpu alleles of the indicated HIV-1 groups and controls. (b) Relationship between Vpu-mediated CD1d down-regulation and iNKT cell activation was assessed using linear regression and Pearson correlation. (c) Effect of expression of different HIV-1 vpu alleles on IFN-γ production by iNKT cells. Blue and green symbols represent subtype B and C Vpu proteins, respectively. Each data point represents one vpu allele and the average value of two experiments performed in at least duplicate.

    Journal: Scientific Reports

    Article Title: Involvement of a C-terminal motif in the interference of primate lentiviral Vpu proteins with CD1d-mediated antigen presentation

    doi: 10.1038/srep09675

    Figure Lengend Snippet: Vpu-mediated down-regulation of CD1d correlates with decreased activation of CD1d-restricted iNKT cells. 293T cells were co-transfected with CD1d and HIV-1 vpu alleles or eGFP control, respectively. At 24 h post transfection, cells were used for analysis of CD1d surface expression levels using flow cytometry and loading with the model lipid antigen αGalCer. The human CD4 + iNKT cell clone HDD3 was co-incubated with the αGalCer-loaded 293T cells in the presence of brefeldin A for 6 h, and subsequently analyzed for IFN-γ production using flow cytometry. iNKT cell IFN-γ production induced by 293T cells co-transfected with CD1d and eGFP control was set to 1, and all samples were calculated relative to control. (a) Representative dot plots showing iNKT cell IFN-γ production induced by 293T cells co-transfected with CD1d and vpu alleles of the indicated HIV-1 groups and controls. (b) Relationship between Vpu-mediated CD1d down-regulation and iNKT cell activation was assessed using linear regression and Pearson correlation. (c) Effect of expression of different HIV-1 vpu alleles on IFN-γ production by iNKT cells. Blue and green symbols represent subtype B and C Vpu proteins, respectively. Each data point represents one vpu allele and the average value of two experiments performed in at least duplicate.

    Article Snippet: iNKT cell activation assay 293T cells were co-transfected with Vpu and CD1d expression vectors and 24 h post transfection loaded with 100 ng/mL αGalCer (KRN7000; Biomol International) for 2 h. 293T cells were then co-incubated with the human CD4+ iNKT cell clone HDD3 at a 1:2 ratio in the presence of brefeldin A (GolgiPLUG; 2 mg/mL; BD Biosciences) for 6 h. To assess iNKT cell IFN-γ production using flow cytometry, cells were stained with anti-CD3-PerCP (clone SK7; BD Biosciences) followed by saponin permeabilization and intracellular staining with anti-IFN-γ-APC (clone 25723.11; BD Biosciences).

    Techniques: Activation Assay, Transfection, Expressing, Flow Cytometry, Cytometry, Incubation

    Vpu proteins of HIV-1 groups M, O and P and their SIV precursors down-regulate CD1d. 293T cells were co-transfected with human CD1d or CD4 and vpu alleles derived from group M, N, O and P viruses and related SIVs, or eGFP control, respectively. CD1d and CD4 surface expression levels were analyzed 24 h post transfection using flow cytometry. Down-regulation was calculated by comparing CD1d and CD4 surface MFIs in cells expressing Vpu/eGFP and the eGFP control. Each symbol represents one vpu allele and the average value of at least three experiments. Horizontal lines indicate average reduction of surface expression of the respective group of vpu alleles. (a) Histograms demonstrating the effect of representative HIV-1 group M, N, O and P Vpu proteins on CD1d surface expression. Blue lines indicate cells expressing eGFP control plasmid; red lines, Vpu/eGFP expressing cells; black lines, non-transfected controls. Figures indicate CD1d MFI values in the absence (blue) or presence (red) of Vpu. (b, c) Reduction of CD1d and CD4 surface expression by HIV-1 and SIV Vpu proteins. Statistical analysis was done using GraphPad Prism software and Kruskal-Wallis test with Dunn's multiple comparisons test. (d, e) As in b, c but HIV-1 group M Vpu proteins divided into subtypes are shown. Statistical analysis was done using an unpaired t-test.

    Journal: Scientific Reports

    Article Title: Involvement of a C-terminal motif in the interference of primate lentiviral Vpu proteins with CD1d-mediated antigen presentation

    doi: 10.1038/srep09675

    Figure Lengend Snippet: Vpu proteins of HIV-1 groups M, O and P and their SIV precursors down-regulate CD1d. 293T cells were co-transfected with human CD1d or CD4 and vpu alleles derived from group M, N, O and P viruses and related SIVs, or eGFP control, respectively. CD1d and CD4 surface expression levels were analyzed 24 h post transfection using flow cytometry. Down-regulation was calculated by comparing CD1d and CD4 surface MFIs in cells expressing Vpu/eGFP and the eGFP control. Each symbol represents one vpu allele and the average value of at least three experiments. Horizontal lines indicate average reduction of surface expression of the respective group of vpu alleles. (a) Histograms demonstrating the effect of representative HIV-1 group M, N, O and P Vpu proteins on CD1d surface expression. Blue lines indicate cells expressing eGFP control plasmid; red lines, Vpu/eGFP expressing cells; black lines, non-transfected controls. Figures indicate CD1d MFI values in the absence (blue) or presence (red) of Vpu. (b, c) Reduction of CD1d and CD4 surface expression by HIV-1 and SIV Vpu proteins. Statistical analysis was done using GraphPad Prism software and Kruskal-Wallis test with Dunn's multiple comparisons test. (d, e) As in b, c but HIV-1 group M Vpu proteins divided into subtypes are shown. Statistical analysis was done using an unpaired t-test.

    Article Snippet: iNKT cell activation assay 293T cells were co-transfected with Vpu and CD1d expression vectors and 24 h post transfection loaded with 100 ng/mL αGalCer (KRN7000; Biomol International) for 2 h. 293T cells were then co-incubated with the human CD4+ iNKT cell clone HDD3 at a 1:2 ratio in the presence of brefeldin A (GolgiPLUG; 2 mg/mL; BD Biosciences) for 6 h. To assess iNKT cell IFN-γ production using flow cytometry, cells were stained with anti-CD3-PerCP (clone SK7; BD Biosciences) followed by saponin permeabilization and intracellular staining with anti-IFN-γ-APC (clone 25723.11; BD Biosciences).

    Techniques: Transfection, Derivative Assay, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Software

    CD1d down-regulation by chimeras between HIV-1 group M subtype B and C Vpu proteins. (a) Schematic representation of WITO.c (BBB; group M subtype B) and ZM247F (CCC; group M subtype C) Vpu chimeras. The TMD, the cytoplasmic domain, α-helical domains 1 and 2, the overlapping putative YxxΦ and [D/E]xxx[L/I] sorting motifs, and the DSGxxS β-TrCP binding site are highlighted. x and Φ correspond to variable and hydrophobic amino acids, respectively. (b, c) 293T cells were co-transfected with CD1d or CD4 and the indicated parental or chimeric Vpu constructs, or eGFP control, respectively. CD1d and CD4 cell surface expression was analyzed by flow cytometry 24 h post transfection, and down-regulation calculated. Average values from at least 3 independent experiments performed in duplicates (±SEM) are shown. Statistical analysis was done using GraphPad Prism software and one-way ANOVA with Tukey's multiple comparisons test.

    Journal: Scientific Reports

    Article Title: Involvement of a C-terminal motif in the interference of primate lentiviral Vpu proteins with CD1d-mediated antigen presentation

    doi: 10.1038/srep09675

    Figure Lengend Snippet: CD1d down-regulation by chimeras between HIV-1 group M subtype B and C Vpu proteins. (a) Schematic representation of WITO.c (BBB; group M subtype B) and ZM247F (CCC; group M subtype C) Vpu chimeras. The TMD, the cytoplasmic domain, α-helical domains 1 and 2, the overlapping putative YxxΦ and [D/E]xxx[L/I] sorting motifs, and the DSGxxS β-TrCP binding site are highlighted. x and Φ correspond to variable and hydrophobic amino acids, respectively. (b, c) 293T cells were co-transfected with CD1d or CD4 and the indicated parental or chimeric Vpu constructs, or eGFP control, respectively. CD1d and CD4 cell surface expression was analyzed by flow cytometry 24 h post transfection, and down-regulation calculated. Average values from at least 3 independent experiments performed in duplicates (±SEM) are shown. Statistical analysis was done using GraphPad Prism software and one-way ANOVA with Tukey's multiple comparisons test.

    Article Snippet: iNKT cell activation assay 293T cells were co-transfected with Vpu and CD1d expression vectors and 24 h post transfection loaded with 100 ng/mL αGalCer (KRN7000; Biomol International) for 2 h. 293T cells were then co-incubated with the human CD4+ iNKT cell clone HDD3 at a 1:2 ratio in the presence of brefeldin A (GolgiPLUG; 2 mg/mL; BD Biosciences) for 6 h. To assess iNKT cell IFN-γ production using flow cytometry, cells were stained with anti-CD3-PerCP (clone SK7; BD Biosciences) followed by saponin permeabilization and intracellular staining with anti-IFN-γ-APC (clone 25723.11; BD Biosciences).

    Techniques: Countercurrent Chromatography, Binding Assay, Transfection, Construct, Expressing, Flow Cytometry, Cytometry, Software

    Determinants at the C-terminus of Vpu contribute to CD1d down-regulation. (a) 293T-CD1d cells were transfected with the indicated non-overlapping triple-alanine mutants of the WITO Vpu C-terminus. 24 h post transfection, cells were surface stained with anti-CD1d antibody and analyzed by flow cytometry. Average values from at least 3 independent experiments preformed in duplicates (±SD) are shown. Statistical analysis was done using GraphPad Prism software and one-way ANOVA with Dunnett's multiple comparisons test. (b) Vpu amino acid sequences of subtype B (n > 5500) and subtype C (n > 3000) were retrieved from the HIV sequence database ( www.hiv.lanl.gov ) and the C-terminal 22 residues of the sequences analyzed using WebLogo 3.0 software (weblogo.threeplusone.com/create.cgi). Colour coding of the amino acids represents hydrophobicity; blue, hydrophilic; green, neutral; black, hydrophobic. Conserved motifs are highlighted.

    Journal: Scientific Reports

    Article Title: Involvement of a C-terminal motif in the interference of primate lentiviral Vpu proteins with CD1d-mediated antigen presentation

    doi: 10.1038/srep09675

    Figure Lengend Snippet: Determinants at the C-terminus of Vpu contribute to CD1d down-regulation. (a) 293T-CD1d cells were transfected with the indicated non-overlapping triple-alanine mutants of the WITO Vpu C-terminus. 24 h post transfection, cells were surface stained with anti-CD1d antibody and analyzed by flow cytometry. Average values from at least 3 independent experiments preformed in duplicates (±SD) are shown. Statistical analysis was done using GraphPad Prism software and one-way ANOVA with Dunnett's multiple comparisons test. (b) Vpu amino acid sequences of subtype B (n > 5500) and subtype C (n > 3000) were retrieved from the HIV sequence database ( www.hiv.lanl.gov ) and the C-terminal 22 residues of the sequences analyzed using WebLogo 3.0 software (weblogo.threeplusone.com/create.cgi). Colour coding of the amino acids represents hydrophobicity; blue, hydrophilic; green, neutral; black, hydrophobic. Conserved motifs are highlighted.

    Article Snippet: iNKT cell activation assay 293T cells were co-transfected with Vpu and CD1d expression vectors and 24 h post transfection loaded with 100 ng/mL αGalCer (KRN7000; Biomol International) for 2 h. 293T cells were then co-incubated with the human CD4+ iNKT cell clone HDD3 at a 1:2 ratio in the presence of brefeldin A (GolgiPLUG; 2 mg/mL; BD Biosciences) for 6 h. To assess iNKT cell IFN-γ production using flow cytometry, cells were stained with anti-CD3-PerCP (clone SK7; BD Biosciences) followed by saponin permeabilization and intracellular staining with anti-IFN-γ-APC (clone 25723.11; BD Biosciences).

    Techniques: Transfection, Staining, Flow Cytometry, Cytometry, Software, Sequencing

    The conserved APW motif of subtype B Vpu proteins is involved in CD1d down-regulation. (a, b) 293T cells were co-transfected with CD1d or CD4 and the indicated WITO and ZM247F Vpu deletion and amino acid exchange mutants, respectively. CD1d and CD4 cell surface expression was analyzed by flow cytometry 24 h post transfection, and down-regulation calculated. Average values from at least 3 independent experiments preformed in duplicates (±SD) are shown. Statistical analysis was done using a Kruskal-Wallis test with Dunn's multiple comparisons test. (c) Expression analysis of parental and mutant Vpu proteins by Western blot. 293T cells were transfected with the indicated eGFP-tagged Vpu proteins, and 24 h post transfection cell lysates were prepared, separated on a 12% SDS–polyacrylamide gel electrophoresis gel and transferred onto a nitrocellulose membrane. The membrane was probed with anti-GFP and anti-actin antibodies. Blots have been cropped; for full-size uncropped blots see Supplementary Fig S6 . (d) Human monocyte-derived dendritic cells were infected with the indicated VSV-G pseudotyped HIV-1 viruses. 7 days post infection, cells were surface stained with anti-CD1d and anti-CD4 antibodies followed by permeabilization, anti-p24 staining and flow cytometry analysis. Down-regulation was calculated by comparing CD1d and CD4 MFIs on p24 negative and positive cells; down-regulation by WITO WT was set to 100%. Data show experiments with MDDCs generated from 3 different donors. Error bars represent SD. (e) 293T cells were transfected with the indicated eGFP-tagged Vpu constructs or eGFP control, respectively. 24 h post transfection, cells were fixed and examined by fluorescence microscopy. eGFP signal intensities were measured along the indicated lines traversing the cells using NIS-Elements A1R software. Vertical dashed lines in the intensity plots indicate cell edges. For all images, contrast and brightness were changed for visualization purposes throughout the entire image. Images shown are representative of 2 independent experiments. Scale bars, 10 μm.

    Journal: Scientific Reports

    Article Title: Involvement of a C-terminal motif in the interference of primate lentiviral Vpu proteins with CD1d-mediated antigen presentation

    doi: 10.1038/srep09675

    Figure Lengend Snippet: The conserved APW motif of subtype B Vpu proteins is involved in CD1d down-regulation. (a, b) 293T cells were co-transfected with CD1d or CD4 and the indicated WITO and ZM247F Vpu deletion and amino acid exchange mutants, respectively. CD1d and CD4 cell surface expression was analyzed by flow cytometry 24 h post transfection, and down-regulation calculated. Average values from at least 3 independent experiments preformed in duplicates (±SD) are shown. Statistical analysis was done using a Kruskal-Wallis test with Dunn's multiple comparisons test. (c) Expression analysis of parental and mutant Vpu proteins by Western blot. 293T cells were transfected with the indicated eGFP-tagged Vpu proteins, and 24 h post transfection cell lysates were prepared, separated on a 12% SDS–polyacrylamide gel electrophoresis gel and transferred onto a nitrocellulose membrane. The membrane was probed with anti-GFP and anti-actin antibodies. Blots have been cropped; for full-size uncropped blots see Supplementary Fig S6 . (d) Human monocyte-derived dendritic cells were infected with the indicated VSV-G pseudotyped HIV-1 viruses. 7 days post infection, cells were surface stained with anti-CD1d and anti-CD4 antibodies followed by permeabilization, anti-p24 staining and flow cytometry analysis. Down-regulation was calculated by comparing CD1d and CD4 MFIs on p24 negative and positive cells; down-regulation by WITO WT was set to 100%. Data show experiments with MDDCs generated from 3 different donors. Error bars represent SD. (e) 293T cells were transfected with the indicated eGFP-tagged Vpu constructs or eGFP control, respectively. 24 h post transfection, cells were fixed and examined by fluorescence microscopy. eGFP signal intensities were measured along the indicated lines traversing the cells using NIS-Elements A1R software. Vertical dashed lines in the intensity plots indicate cell edges. For all images, contrast and brightness were changed for visualization purposes throughout the entire image. Images shown are representative of 2 independent experiments. Scale bars, 10 μm.

    Article Snippet: iNKT cell activation assay 293T cells were co-transfected with Vpu and CD1d expression vectors and 24 h post transfection loaded with 100 ng/mL αGalCer (KRN7000; Biomol International) for 2 h. 293T cells were then co-incubated with the human CD4+ iNKT cell clone HDD3 at a 1:2 ratio in the presence of brefeldin A (GolgiPLUG; 2 mg/mL; BD Biosciences) for 6 h. To assess iNKT cell IFN-γ production using flow cytometry, cells were stained with anti-CD3-PerCP (clone SK7; BD Biosciences) followed by saponin permeabilization and intracellular staining with anti-IFN-γ-APC (clone 25723.11; BD Biosciences).

    Techniques: Transfection, Expressing, Flow Cytometry, Cytometry, Mutagenesis, Western Blot, Polyacrylamide Gel Electrophoresis, Derivative Assay, Infection, Staining, Generated, Construct, Fluorescence, Microscopy, Software

    Phosphatidylinositol-3-kinase (PI3K) and Akt are downstream of interferon (IFN) promoter stimulator-1 (IPS-1) activating IFN regulatory factor 3 (IRF3) activation and IFN- β expression. (a–d) 293T cells were transfected with an expression

    Journal: Immunology

    Article Title: Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1

    doi: 10.1111/imm.12373

    Figure Lengend Snippet: Phosphatidylinositol-3-kinase (PI3K) and Akt are downstream of interferon (IFN) promoter stimulator-1 (IPS-1) activating IFN regulatory factor 3 (IRF3) activation and IFN- β expression. (a–d) 293T cells were transfected with an expression

    Article Snippet: Human Akt-specific siRNA used for 293T cells was from Cell Signaling Technology.

    Techniques: Activation Assay, Expressing, Transfection

    Phosphatidylinositol-3-kinase (PI3K) and Akt are required for retinoic acid-inducible gene I (RIG-I) -induced interferon (IFN) regulatory factor 3 (IRF3) activation and IFN- β expression. (a–d) 293T cells were transfected with (a, c) an

    Journal: Immunology

    Article Title: Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1

    doi: 10.1111/imm.12373

    Figure Lengend Snippet: Phosphatidylinositol-3-kinase (PI3K) and Akt are required for retinoic acid-inducible gene I (RIG-I) -induced interferon (IFN) regulatory factor 3 (IRF3) activation and IFN- β expression. (a–d) 293T cells were transfected with (a, c) an

    Article Snippet: Human Akt-specific siRNA used for 293T cells was from Cell Signaling Technology.

    Techniques: Activation Assay, Expressing, Transfection

    PNDA-3 inhibits periostin-dependent signaling. ( a ) Schematic representation of the full-length periostin and deletion constructs used in this study. ( b ) Biotinylated PNDA-3 was mixed with cell lysates from 293T cells overexpressing mutant periostins,

    Journal: Molecular Therapy

    Article Title: Periostin-binding DNA Aptamer Inhibits Breast Cancer Growth and Metastasis

    doi: 10.1038/mt.2013.30

    Figure Lengend Snippet: PNDA-3 inhibits periostin-dependent signaling. ( a ) Schematic representation of the full-length periostin and deletion constructs used in this study. ( b ) Biotinylated PNDA-3 was mixed with cell lysates from 293T cells overexpressing mutant periostins,

    Article Snippet: The MDA-MB-231, 4T1, and 293T cells were cultured in Dulbecco's modified Eagle's medium (Lonza) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (Gibco).

    Techniques: Construct, Mutagenesis

    CDK9 controls Ser2P and is essential for activator-driven transcription. (A) ChIP profile of CTD Ser5P, and (B) Ser2P across the p21 and GAPDH loci in the absence or presence of 067. Lower graphs show relative levels of CTD modifications after normalizing to RNAPII. (C) ChIP profile of CDK9 (and, for comparison, of Ser2P). (D) Immunoblot (IB) analysis of RNAPII and bulk levels of Ser2P/5P/7P in control, CDK9 and CDK12 knockdown cells. Band intensities of CTD modifications were determined using ImageJ and normalized to RNAPII in the control sample. (E) Effect of CDK9 or CDK12 knockdown on p21 inducibility was determined as in Fig 3A . (F) Nuclear extracts (NE) from 293T cells were immunodepleted (□) with non-specific IgG or the indicated antibodies and analyzed by immunoblotting (IB). (G) Depleted extracts were used in in vitro transcription reactions together with recombinant Gal4-VP16 activator and a responsive PCR template containing the Adenovirus major late promoter (AdML). Transcription elongation continued for 5 or 12 minutes, and radioactively labeled transcripts (tx) were visualized by autoradiography.

    Journal: PLoS ONE

    Article Title: The Establishment of a Hyperactive Structure Allows the Tumour Suppressor Protein p53 to Function through P-TEFb during Limited CDK9 Kinase Inhibition

    doi: 10.1371/journal.pone.0146648

    Figure Lengend Snippet: CDK9 controls Ser2P and is essential for activator-driven transcription. (A) ChIP profile of CTD Ser5P, and (B) Ser2P across the p21 and GAPDH loci in the absence or presence of 067. Lower graphs show relative levels of CTD modifications after normalizing to RNAPII. (C) ChIP profile of CDK9 (and, for comparison, of Ser2P). (D) Immunoblot (IB) analysis of RNAPII and bulk levels of Ser2P/5P/7P in control, CDK9 and CDK12 knockdown cells. Band intensities of CTD modifications were determined using ImageJ and normalized to RNAPII in the control sample. (E) Effect of CDK9 or CDK12 knockdown on p21 inducibility was determined as in Fig 3A . (F) Nuclear extracts (NE) from 293T cells were immunodepleted (□) with non-specific IgG or the indicated antibodies and analyzed by immunoblotting (IB). (G) Depleted extracts were used in in vitro transcription reactions together with recombinant Gal4-VP16 activator and a responsive PCR template containing the Adenovirus major late promoter (AdML). Transcription elongation continued for 5 or 12 minutes, and radioactively labeled transcripts (tx) were visualized by autoradiography.

    Article Snippet: Cells MCF7, A549, HeLa and 293T cells were obtained from DSMZ (Braunschweig, Germany) or ATCC (Manassas, VA, USA) and cultivated in DMEM medium supplemented with L-glutamine (2 mM), penicillin-streptomycin (100 units/ml-100 μg/ml; all from Life Technologies, Darmstadt, Germany) and 10% FBS (FBS Gold; GE Healthcare).

    Techniques: Chromatin Immunoprecipitation, In Vitro, Recombinant, Polymerase Chain Reaction, Labeling, Autoradiography

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

    Journal:

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

    doi: 10.1074/jbc.M610623200

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

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

    Techniques:

    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