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  • 99
    Promega posttransfection
    The Cal/09 NS1 protein is unable to block general gene expression in human or swine cells. Human 293T (A) or swine PK-15 (C) cells were cotransfected with a pCAGGS expression plasmid encoding the indicated NS1 protein (or GST) together with a constitutively active Renilla luciferase plasmid. Luciferase activity was determined 24 h <t>posttransfection.</t> Results show the means and standard deviations of triplicate values (normalized to GST [A] or PR/34 NS1 [C]) obtained in a single experiment and are representative of results of two independent experiments. (B) Western blot analysis of lysates from panel A. (D) Western blot analysis of lysates from panel C. NS1 and GST were detected using a polyclonal rabbit anti-serum raised against a fusion protein of GST and the N-terminal RNA binding domain of NS1. Tubulin acted as a loading control. Molecular mass markers (in kilodaltons) are indicated to the right.
    Posttransfection, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 6466 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson posttransfection
    The mutant TAp63γ proteins defective in DNA binding or in transactivation are nuclear and stable. (A) H1299 cells were transfected with an indicated p53 or TAp63γ expression plasmid. Equal amounts of nuclear extracts were subjected to EMSA using a 32 P-labeled p53 oligonucleotide (Oligo) (top panel). Cold competition was performed using either the wild-type (wt) p53 oligonucleotide (lanes 3 and 6) or a mutant (mt) p53 oligonucleotide (lanes 4 and 7). Expression of ectopically expressed p53 or p63 proteins was shown by Western blot analysis for p53, p63, and actin (bottom panel). V, vector. (B) Dose effects of wild-type or mutant TAp63γ on transactivation activity. Saos-2 cells were transfected with the indicated amounts of wild-type or mutant TAp63γ [TAp63γ(FWL-A) or TAp63γ(R304W)] in the presence of Bax-Luciferase reporter and pCMV-β-galactosidase plasmids. Equal amounts of total proteins (20 μg) were subjected to Western blot analysis for p63 and actin (top panel) or to assays measuring luciferase and β-galactosidase activities. The luciferase activity was normalized to β-galactosidase activity and reported as an increase in activation ( n -fold) (mean ± standard deviation [error bar]) (bottom panel). V, vector. (C) U2-OS cells were cotransfected with wild-type or mutant TAp63γ and GFP expression plasmids. Thirty-six hours <t>posttransfection,</t> cells were fixed and subjected to immunohistochemical staining with p63-specific antibody (4A4). Nuclei were costained with DAPI. Coexpressed GFP is shown (magnification of ×400).
    Posttransfection, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 658 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher posttransfection
    The mutant TAp63γ proteins defective in DNA binding or in transactivation are nuclear and stable. (A) H1299 cells were transfected with an indicated p53 or TAp63γ expression plasmid. Equal amounts of nuclear extracts were subjected to EMSA using a 32 P-labeled p53 oligonucleotide (Oligo) (top panel). Cold competition was performed using either the wild-type (wt) p53 oligonucleotide (lanes 3 and 6) or a mutant (mt) p53 oligonucleotide (lanes 4 and 7). Expression of ectopically expressed p53 or p63 proteins was shown by Western blot analysis for p53, p63, and actin (bottom panel). V, vector. (B) Dose effects of wild-type or mutant TAp63γ on transactivation activity. Saos-2 cells were transfected with the indicated amounts of wild-type or mutant TAp63γ [TAp63γ(FWL-A) or TAp63γ(R304W)] in the presence of Bax-Luciferase reporter and pCMV-β-galactosidase plasmids. Equal amounts of total proteins (20 μg) were subjected to Western blot analysis for p63 and actin (top panel) or to assays measuring luciferase and β-galactosidase activities. The luciferase activity was normalized to β-galactosidase activity and reported as an increase in activation ( n -fold) (mean ± standard deviation [error bar]) (bottom panel). V, vector. (C) U2-OS cells were cotransfected with wild-type or mutant TAp63γ and GFP expression plasmids. Thirty-six hours <t>posttransfection,</t> cells were fixed and subjected to immunohistochemical staining with p63-specific antibody (4A4). Nuclei were costained with DAPI. Coexpressed GFP is shown (magnification of ×400).
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    94
    Millipore posttransfection
    Binding analysis of escape mutants generated against two divergent H3N2 viruses. An escape mutant generated against Scot/74 (emScot/74) revealed one point mutation (R25M) in the HA2 domain, while an escape mutant against Vic/11 (emVic/11) had two point mutations (D19N and T107A). (A) An H3 HA monomer depicted using PyMOL shows the location of the point mutations found on emScot/74 HA (green) and emVic/11 HA (red), where HA1 is indicated in blue and the HA2 is indicated in yellow. The point mutation R25M (green) is located in the middle of the beta-sheet of the HA2 domain (yellow) of Scot/74 HA. The D19N (red) residue change is located at the elbow of the fusion peptide, while the T107A (red) resides in the middle of the long α-helix facing the short α-helix of HA2 (yellow) of Vic/11 HA. (B) 293T cells were transfected with wild-type or escape mutant HA and fixed at 24 h <t>posttransfection.</t> Reactivity was assessed by immunofluorescence using MAb 9H10 or MAb CR8020 (5 μg/ml). Infected mouse serum was used as a positive control, and a pan-H1 stalk MAb, 6F12 (5 μg/ml), was used as a negative control.
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    93
    Beckman Coulter posttransfection
    The gp42 linker mutants are expressed independently of gHgL expression and are not functional in B cell fusion. CHO-KI cells were transiently transfected with 2 µg of the gp42 linker mutants either in the presence or absence of gHgL and gB (0.5 µg each) and T7 luciferase (0.8 µg). Eighteen hours <t>posttransfection,</t> 40,000 cells were transferred to each well of a black 96-well plate and overlaid with 40,000 Daudi B cells for fusion assay. Eighteen hours after overlay, cells were washed with phosphate-buffered saline (PBS) and lysed for 10 min with 50 µl of passive lysis buffer (Promega) per well. Luciferase activity was measured with a PerkinElmer Victor plate reader immediately after addition of 50 µl/well of luciferase reagent (Promega). Eighteen hours posttransfection, 80,000 cells were transferred to each well of a clear 96-well plate, and eighteen hours later, surface expression was determined by cELISA using anti-gp42 (3H3), fixation, secondary biotinylated anti-mouse IgG antibody (Sigma), tertiary streptavidin-horseradish peroxidase (GE Healthcare) and TMB (3,3=,5,5=-tetramethylbenzidine) after TMB one-component substrate (BioFX). Absorbance readings were taken at 380 nm using a PerkinElmer Victor plate reader. Binding was normalized to wild-type gp42 binding levels, which were set at 100%. Data shown are representative of the results from three independent experiments.
    Posttransfection, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 93/100, based on 501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim posttransfection
    Mapping of the activation domain of Luman by deletion analysis. On the left is a schematic representation of the structure of the Luman protein, in which numbers indicate the positions of the amino acids. The X in the HCF-binding domain of D78A represents a point mutation (alanine replaces the aspartate residue at position 78). Features of the protein discussed in the text are labeled. Luman and its deletion mutants were fused to the GAL4 DBD. The same amount (0.5 μg) of each plasmid was introduced into COS7 cells along with the reporter plasmid, pG5EC (0.5 μg), which has five copies of GAL4 UAS in the promoter region linked to the cat gene. CAT activity was measured by ELISA 48 h <t>posttransfection.</t>
    Posttransfection, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare posttransfection
    Morphology of PA-457-resistant virus particles. Thin-section transmission EM analysis of virions with the indicated mutations produced from HeLa cells cultured without PA-457 or with 5.0 μg/ml PA-457 is shown. Cells were fixed at 48 h <t>posttransfection</t> and were analyzed by EM. (a to c) WT pNL4-3 without (a) or with (b and c) PA-457; (d and e) SP1-A1V without (d) or with (e) PA-457; (f to i) SP1-A3V without PA-457; (j to l) SP1-A3T without PA-457; (m and n) SP1-A3V with PA-457; (o to q) SP1-A3T with PA-457; (r and s) CA-G225S/SP1-A3V without (r) or with (s) PA-457. Bars, 200 nm.
    Posttransfection, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 564 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Carl Roth GmbH posttransfection
    Morphology of PA-457-resistant virus particles. Thin-section transmission EM analysis of virions with the indicated mutations produced from HeLa cells cultured without PA-457 or with 5.0 μg/ml PA-457 is shown. Cells were fixed at 48 h <t>posttransfection</t> and were analyzed by EM. (a to c) WT pNL4-3 without (a) or with (b and c) PA-457; (d and e) SP1-A1V without (d) or with (e) PA-457; (f to i) SP1-A3V without PA-457; (j to l) SP1-A3T without PA-457; (m and n) SP1-A3V with PA-457; (o to q) SP1-A3T with PA-457; (r and s) CA-G225S/SP1-A3V without (r) or with (s) PA-457. Bars, 200 nm.
    Posttransfection, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 91/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sarstedt posttransfection
    Morphology of PA-457-resistant virus particles. Thin-section transmission EM analysis of virions with the indicated mutations produced from HeLa cells cultured without PA-457 or with 5.0 μg/ml PA-457 is shown. Cells were fixed at 48 h <t>posttransfection</t> and were analyzed by EM. (a to c) WT pNL4-3 without (a) or with (b and c) PA-457; (d and e) SP1-A1V without (d) or with (e) PA-457; (f to i) SP1-A3V without PA-457; (j to l) SP1-A3T without PA-457; (m and n) SP1-A3V with PA-457; (o to q) SP1-A3T with PA-457; (r and s) CA-G225S/SP1-A3V without (r) or with (s) PA-457. Bars, 200 nm.
    Posttransfection, supplied by Sarstedt, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioVision posttransfection
    Morphology of PA-457-resistant virus particles. Thin-section transmission EM analysis of virions with the indicated mutations produced from HeLa cells cultured without PA-457 or with 5.0 μg/ml PA-457 is shown. Cells were fixed at 48 h <t>posttransfection</t> and were analyzed by EM. (a to c) WT pNL4-3 without (a) or with (b and c) PA-457; (d and e) SP1-A1V without (d) or with (e) PA-457; (f to i) SP1-A3V without PA-457; (j to l) SP1-A3T without PA-457; (m and n) SP1-A3V with PA-457; (o to q) SP1-A3T with PA-457; (r and s) CA-G225S/SP1-A3V without (r) or with (s) PA-457. Bars, 200 nm.
    Posttransfection, supplied by BioVision, 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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    93
    Merck KGaA posttransfection
    Morphology of PA-457-resistant virus particles. Thin-section transmission EM analysis of virions with the indicated mutations produced from HeLa cells cultured without PA-457 or with 5.0 μg/ml PA-457 is shown. Cells were fixed at 48 h <t>posttransfection</t> and were analyzed by EM. (a to c) WT pNL4-3 without (a) or with (b and c) PA-457; (d and e) SP1-A1V without (d) or with (e) PA-457; (f to i) SP1-A3V without PA-457; (j to l) SP1-A3T without PA-457; (m and n) SP1-A3V with PA-457; (o to q) SP1-A3T with PA-457; (r and s) CA-G225S/SP1-A3V without (r) or with (s) PA-457. Bars, 200 nm.
    Posttransfection, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology posttransfection
    Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h <t>posttransfection.</t> Six pictures per condition were analyzed with the CellProfiler program to determine percentages of positive cells or mean integrated intensities of stained cells. **, P
    Posttransfection, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences posttransfection
    Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h <t>posttransfection.</t> Six pictures per condition were analyzed with the CellProfiler program to determine percentages of positive cells or mean integrated intensities of stained cells. **, P
    Posttransfection, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FlowJo posttransfection
    Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h <t>posttransfection.</t> Six pictures per condition were analyzed with the CellProfiler program to determine percentages of positive cells or mean integrated intensities of stained cells. **, P
    Posttransfection, supplied by FlowJo, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GraphPad Prism Inc posttransfection
    Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h <t>posttransfection.</t> Six pictures per condition were analyzed with the CellProfiler program to determine percentages of positive cells or mean integrated intensities of stained cells. **, P
    Posttransfection, supplied by GraphPad Prism Inc, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ibidi posttransfection
    Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h <t>posttransfection.</t> Six pictures per condition were analyzed with the CellProfiler program to determine percentages of positive cells or mean integrated intensities of stained cells. **, P
    Posttransfection, supplied by ibidi, 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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    92
    Polyplus Transfection posttransfection
    Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h <t>posttransfection.</t> Six pictures per condition were analyzed with the CellProfiler program to determine percentages of positive cells or mean integrated intensities of stained cells. **, P
    Posttransfection, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc posttransfection
    IL-6 activates Etk through PI3-kinase. ( A ) Wortmannin (Wort) blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk. At 24 hr <t>posttransfection,</t> the cells were serum starved for 24 hr. The cells were then pretreated with 100 nM wortmannin (+) or dimethyl sulfoxide Mock (−) for 30 min followed by IL-6 treatment for 30 min. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-T7 antibody ( Lower ). The tyrosine phosphorylation of the T7-tagged Etk is indicated. ( B ) A myrislated p110 activates Etk. The CV-1 cells were transfected by pcDNA3-T7-Etk in the presence (+) or absence (−) of pcDNA3-p110 myr. At 24 hr posttransfection, the cells were serum starved for 24 hr. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with anti-phosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ). ( C ) A dominant negative p85 blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk (wild type) or pcDNA3-T7-EtkΔPH(deletion of PH domain) in the presence (+) or absence (−) of pcDNA3-p85DN. At 24 hr posttransfection, the cells were serum starved for 24 hr followed by IL-6 treatment for 30 min. The cell extracts from each treatment were subjected to immunoprecipitation by anti-T7 antibody, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ).
    Posttransfection, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Avantor posttransfection
    IL-6 activates Etk through PI3-kinase. ( A ) Wortmannin (Wort) blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk. At 24 hr <t>posttransfection,</t> the cells were serum starved for 24 hr. The cells were then pretreated with 100 nM wortmannin (+) or dimethyl sulfoxide Mock (−) for 30 min followed by IL-6 treatment for 30 min. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-T7 antibody ( Lower ). The tyrosine phosphorylation of the T7-tagged Etk is indicated. ( B ) A myrislated p110 activates Etk. The CV-1 cells were transfected by pcDNA3-T7-Etk in the presence (+) or absence (−) of pcDNA3-p110 myr. At 24 hr posttransfection, the cells were serum starved for 24 hr. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with anti-phosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ). ( C ) A dominant negative p85 blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk (wild type) or pcDNA3-T7-EtkΔPH(deletion of PH domain) in the presence (+) or absence (−) of pcDNA3-p85DN. At 24 hr posttransfection, the cells were serum starved for 24 hr followed by IL-6 treatment for 30 min. The cell extracts from each treatment were subjected to immunoprecipitation by anti-T7 antibody, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ).
    Posttransfection, supplied by Avantor, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    KEYENCE posttransfection
    IL-6 activates Etk through PI3-kinase. ( A ) Wortmannin (Wort) blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk. At 24 hr <t>posttransfection,</t> the cells were serum starved for 24 hr. The cells were then pretreated with 100 nM wortmannin (+) or dimethyl sulfoxide Mock (−) for 30 min followed by IL-6 treatment for 30 min. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-T7 antibody ( Lower ). The tyrosine phosphorylation of the T7-tagged Etk is indicated. ( B ) A myrislated p110 activates Etk. The CV-1 cells were transfected by pcDNA3-T7-Etk in the presence (+) or absence (−) of pcDNA3-p110 myr. At 24 hr posttransfection, the cells were serum starved for 24 hr. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with anti-phosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ). ( C ) A dominant negative p85 blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk (wild type) or pcDNA3-T7-EtkΔPH(deletion of PH domain) in the presence (+) or absence (−) of pcDNA3-p85DN. At 24 hr posttransfection, the cells were serum starved for 24 hr followed by IL-6 treatment for 30 min. The cell extracts from each treatment were subjected to immunoprecipitation by anti-T7 antibody, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ).
    Posttransfection, supplied by KEYENCE, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Novoprotein posttransfection
    NRIP blocks the polyubiquitination and phosphorylation of HPV-16 E2 through its CaM binding domain. (A) The expression vectors for HA-tagged ubiquitin, EGFP-16E2, NRIP-FLAG, and NRIPΔIQ-FLAG were transfected into 293 cells. At 40 h <t>posttransfection,</t>
    Posttransfection, supplied by Novoprotein, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Sysmex Corporation posttransfection
    NRIP blocks the polyubiquitination and phosphorylation of HPV-16 E2 through its CaM binding domain. (A) The expression vectors for HA-tagged ubiquitin, EGFP-16E2, NRIP-FLAG, and NRIPΔIQ-FLAG were transfected into 293 cells. At 40 h <t>posttransfection,</t>
    Posttransfection, supplied by Sysmex Corporation, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PerkinElmer posttransfection
    NRIP blocks the polyubiquitination and phosphorylation of HPV-16 E2 through its CaM binding domain. (A) The expression vectors for HA-tagged ubiquitin, EGFP-16E2, NRIP-FLAG, and NRIPΔIQ-FLAG were transfected into 293 cells. At 40 h <t>posttransfection,</t>
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    NRIP blocks the polyubiquitination and phosphorylation of HPV-16 E2 through its CaM binding domain. (A) The expression vectors for HA-tagged ubiquitin, EGFP-16E2, NRIP-FLAG, and NRIPΔIQ-FLAG were transfected into 293 cells. At 40 h <t>posttransfection,</t>
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    Image Search Results


    The Cal/09 NS1 protein is unable to block general gene expression in human or swine cells. Human 293T (A) or swine PK-15 (C) cells were cotransfected with a pCAGGS expression plasmid encoding the indicated NS1 protein (or GST) together with a constitutively active Renilla luciferase plasmid. Luciferase activity was determined 24 h posttransfection. Results show the means and standard deviations of triplicate values (normalized to GST [A] or PR/34 NS1 [C]) obtained in a single experiment and are representative of results of two independent experiments. (B) Western blot analysis of lysates from panel A. (D) Western blot analysis of lysates from panel C. NS1 and GST were detected using a polyclonal rabbit anti-serum raised against a fusion protein of GST and the N-terminal RNA binding domain of NS1. Tubulin acted as a loading control. Molecular mass markers (in kilodaltons) are indicated to the right.

    Journal: Journal of Virology

    Article Title: Inefficient Control of Host Gene Expression by the 2009 Pandemic H1N1 Influenza A Virus NS1 Protein ▿

    doi: 10.1128/JVI.00081-10

    Figure Lengend Snippet: The Cal/09 NS1 protein is unable to block general gene expression in human or swine cells. Human 293T (A) or swine PK-15 (C) cells were cotransfected with a pCAGGS expression plasmid encoding the indicated NS1 protein (or GST) together with a constitutively active Renilla luciferase plasmid. Luciferase activity was determined 24 h posttransfection. Results show the means and standard deviations of triplicate values (normalized to GST [A] or PR/34 NS1 [C]) obtained in a single experiment and are representative of results of two independent experiments. (B) Western blot analysis of lysates from panel A. (D) Western blot analysis of lysates from panel C. NS1 and GST were detected using a polyclonal rabbit anti-serum raised against a fusion protein of GST and the N-terminal RNA binding domain of NS1. Tubulin acted as a loading control. Molecular mass markers (in kilodaltons) are indicated to the right.

    Article Snippet: Expression of REN-Luc activity was measured 24 h posttransfection as directed by the manufacturer (Promega, WI).

    Techniques: Blocking Assay, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, RNA Binding Assay

    The mutant TAp63γ proteins defective in DNA binding or in transactivation are nuclear and stable. (A) H1299 cells were transfected with an indicated p53 or TAp63γ expression plasmid. Equal amounts of nuclear extracts were subjected to EMSA using a 32 P-labeled p53 oligonucleotide (Oligo) (top panel). Cold competition was performed using either the wild-type (wt) p53 oligonucleotide (lanes 3 and 6) or a mutant (mt) p53 oligonucleotide (lanes 4 and 7). Expression of ectopically expressed p53 or p63 proteins was shown by Western blot analysis for p53, p63, and actin (bottom panel). V, vector. (B) Dose effects of wild-type or mutant TAp63γ on transactivation activity. Saos-2 cells were transfected with the indicated amounts of wild-type or mutant TAp63γ [TAp63γ(FWL-A) or TAp63γ(R304W)] in the presence of Bax-Luciferase reporter and pCMV-β-galactosidase plasmids. Equal amounts of total proteins (20 μg) were subjected to Western blot analysis for p63 and actin (top panel) or to assays measuring luciferase and β-galactosidase activities. The luciferase activity was normalized to β-galactosidase activity and reported as an increase in activation ( n -fold) (mean ± standard deviation [error bar]) (bottom panel). V, vector. (C) U2-OS cells were cotransfected with wild-type or mutant TAp63γ and GFP expression plasmids. Thirty-six hours posttransfection, cells were fixed and subjected to immunohistochemical staining with p63-specific antibody (4A4). Nuclei were costained with DAPI. Coexpressed GFP is shown (magnification of ×400).

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Binding and Transactivation Activities Are Essential for TAp63 Protein Degradation

    doi: 10.1128/MCB.25.14.6154-6164.2005

    Figure Lengend Snippet: The mutant TAp63γ proteins defective in DNA binding or in transactivation are nuclear and stable. (A) H1299 cells were transfected with an indicated p53 or TAp63γ expression plasmid. Equal amounts of nuclear extracts were subjected to EMSA using a 32 P-labeled p53 oligonucleotide (Oligo) (top panel). Cold competition was performed using either the wild-type (wt) p53 oligonucleotide (lanes 3 and 6) or a mutant (mt) p53 oligonucleotide (lanes 4 and 7). Expression of ectopically expressed p53 or p63 proteins was shown by Western blot analysis for p53, p63, and actin (bottom panel). V, vector. (B) Dose effects of wild-type or mutant TAp63γ on transactivation activity. Saos-2 cells were transfected with the indicated amounts of wild-type or mutant TAp63γ [TAp63γ(FWL-A) or TAp63γ(R304W)] in the presence of Bax-Luciferase reporter and pCMV-β-galactosidase plasmids. Equal amounts of total proteins (20 μg) were subjected to Western blot analysis for p63 and actin (top panel) or to assays measuring luciferase and β-galactosidase activities. The luciferase activity was normalized to β-galactosidase activity and reported as an increase in activation ( n -fold) (mean ± standard deviation [error bar]) (bottom panel). V, vector. (C) U2-OS cells were cotransfected with wild-type or mutant TAp63γ and GFP expression plasmids. Thirty-six hours posttransfection, cells were fixed and subjected to immunohistochemical staining with p63-specific antibody (4A4). Nuclei were costained with DAPI. Coexpressed GFP is shown (magnification of ×400).

    Article Snippet: Thirty-six hours posttransfection, cells were harvested in 1× reporter lysis buffer (BD Biosciences-Pharmingen) and subjected to β-galactosidase assay and luciferase activity assay (BD Biosciences-Pharmingen).

    Techniques: Mutagenesis, Binding Assay, Transfection, Expressing, Plasmid Preparation, Labeling, Western Blot, Activity Assay, Luciferase, Activation Assay, Standard Deviation, Immunohistochemistry, Staining

    Binding analysis of escape mutants generated against two divergent H3N2 viruses. An escape mutant generated against Scot/74 (emScot/74) revealed one point mutation (R25M) in the HA2 domain, while an escape mutant against Vic/11 (emVic/11) had two point mutations (D19N and T107A). (A) An H3 HA monomer depicted using PyMOL shows the location of the point mutations found on emScot/74 HA (green) and emVic/11 HA (red), where HA1 is indicated in blue and the HA2 is indicated in yellow. The point mutation R25M (green) is located in the middle of the beta-sheet of the HA2 domain (yellow) of Scot/74 HA. The D19N (red) residue change is located at the elbow of the fusion peptide, while the T107A (red) resides in the middle of the long α-helix facing the short α-helix of HA2 (yellow) of Vic/11 HA. (B) 293T cells were transfected with wild-type or escape mutant HA and fixed at 24 h posttransfection. Reactivity was assessed by immunofluorescence using MAb 9H10 or MAb CR8020 (5 μg/ml). Infected mouse serum was used as a positive control, and a pan-H1 stalk MAb, 6F12 (5 μg/ml), was used as a negative control.

    Journal: Journal of Virology

    Article Title: Characterization of a Broadly Neutralizing Monoclonal Antibody That Targets the Fusion Domain of Group 2 Influenza A Virus Hemagglutinin

    doi: 10.1128/JVI.02289-14

    Figure Lengend Snippet: Binding analysis of escape mutants generated against two divergent H3N2 viruses. An escape mutant generated against Scot/74 (emScot/74) revealed one point mutation (R25M) in the HA2 domain, while an escape mutant against Vic/11 (emVic/11) had two point mutations (D19N and T107A). (A) An H3 HA monomer depicted using PyMOL shows the location of the point mutations found on emScot/74 HA (green) and emVic/11 HA (red), where HA1 is indicated in blue and the HA2 is indicated in yellow. The point mutation R25M (green) is located in the middle of the beta-sheet of the HA2 domain (yellow) of Scot/74 HA. The D19N (red) residue change is located at the elbow of the fusion peptide, while the T107A (red) resides in the middle of the long α-helix facing the short α-helix of HA2 (yellow) of Vic/11 HA. (B) 293T cells were transfected with wild-type or escape mutant HA and fixed at 24 h posttransfection. Reactivity was assessed by immunofluorescence using MAb 9H10 or MAb CR8020 (5 μg/ml). Infected mouse serum was used as a positive control, and a pan-H1 stalk MAb, 6F12 (5 μg/ml), was used as a negative control.

    Article Snippet: Cultures were spun down, and the supernatant was harvested at 6 days posttransfection and purified via a Protein G column (Millipore, Inc.).

    Techniques: Binding Assay, Generated, Mutagenesis, Transfection, Immunofluorescence, Infection, Positive Control, Negative Control

    The gp42 linker mutants are expressed independently of gHgL expression and are not functional in B cell fusion. CHO-KI cells were transiently transfected with 2 µg of the gp42 linker mutants either in the presence or absence of gHgL and gB (0.5 µg each) and T7 luciferase (0.8 µg). Eighteen hours posttransfection, 40,000 cells were transferred to each well of a black 96-well plate and overlaid with 40,000 Daudi B cells for fusion assay. Eighteen hours after overlay, cells were washed with phosphate-buffered saline (PBS) and lysed for 10 min with 50 µl of passive lysis buffer (Promega) per well. Luciferase activity was measured with a PerkinElmer Victor plate reader immediately after addition of 50 µl/well of luciferase reagent (Promega). Eighteen hours posttransfection, 80,000 cells were transferred to each well of a clear 96-well plate, and eighteen hours later, surface expression was determined by cELISA using anti-gp42 (3H3), fixation, secondary biotinylated anti-mouse IgG antibody (Sigma), tertiary streptavidin-horseradish peroxidase (GE Healthcare) and TMB (3,3=,5,5=-tetramethylbenzidine) after TMB one-component substrate (BioFX). Absorbance readings were taken at 380 nm using a PerkinElmer Victor plate reader. Binding was normalized to wild-type gp42 binding levels, which were set at 100%. Data shown are representative of the results from three independent experiments.

    Journal: mBio

    Article Title: Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

    doi: 10.1128/mBio.02285-14

    Figure Lengend Snippet: The gp42 linker mutants are expressed independently of gHgL expression and are not functional in B cell fusion. CHO-KI cells were transiently transfected with 2 µg of the gp42 linker mutants either in the presence or absence of gHgL and gB (0.5 µg each) and T7 luciferase (0.8 µg). Eighteen hours posttransfection, 40,000 cells were transferred to each well of a black 96-well plate and overlaid with 40,000 Daudi B cells for fusion assay. Eighteen hours after overlay, cells were washed with phosphate-buffered saline (PBS) and lysed for 10 min with 50 µl of passive lysis buffer (Promega) per well. Luciferase activity was measured with a PerkinElmer Victor plate reader immediately after addition of 50 µl/well of luciferase reagent (Promega). Eighteen hours posttransfection, 80,000 cells were transferred to each well of a clear 96-well plate, and eighteen hours later, surface expression was determined by cELISA using anti-gp42 (3H3), fixation, secondary biotinylated anti-mouse IgG antibody (Sigma), tertiary streptavidin-horseradish peroxidase (GE Healthcare) and TMB (3,3=,5,5=-tetramethylbenzidine) after TMB one-component substrate (BioFX). Absorbance readings were taken at 380 nm using a PerkinElmer Victor plate reader. Binding was normalized to wild-type gp42 binding levels, which were set at 100%. Data shown are representative of the results from three independent experiments.

    Article Snippet: The medium was changed 16 h posttransfection, and 1 h later, the cells were detached with Versene, counted using a Beckman Coulter Z1 particle counter, 37,500 cells were transferred to each well of a 96-well plate, and the total volume was adjusted to 150 µl with complete Ham’s F-12 medium.

    Techniques: Expressing, Functional Assay, Transfection, Luciferase, Single Vesicle Fusion Assay, Lysis, Activity Assay, Binding Assay

    The gp42 linker mutants act in a dominant-negative fashion and compete with wild-type gp42 during B cell fusion. CHO-KI cells were transiently transfected with 0.5 µg of wild-type gp42 and increasing amounts of each of the gp42 linker mutants (0.01 µg, 0.10 µg, and 1.0 µg) indicated by the thickness of the gray wedge at the bottom of the figure in the presence of gH (0.5 µg), gL (0.5 µg), gB (0.5 µg), and T7 polymerase (0.8 µg). Twenty-four hours posttransfection, cells were detached and overlaid with Daudi B cells expressing HLA class II and T7 promoter. Zero, 2 or 20 µl of sgp42 supernatant was added at the time of overlay (white, gray, and black bars). Twenty hours after the overlay, fusion activity was assessed by the addition of passive lysis buffer, followed by the addition of luciferase substrate. Luciferase activity was measured with a PerkinElmer Victor plate reader. Data shown are results representative of the results of three independent experiments. Error bars represent standard deviations for the normalized values.

    Journal: mBio

    Article Title: Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

    doi: 10.1128/mBio.02285-14

    Figure Lengend Snippet: The gp42 linker mutants act in a dominant-negative fashion and compete with wild-type gp42 during B cell fusion. CHO-KI cells were transiently transfected with 0.5 µg of wild-type gp42 and increasing amounts of each of the gp42 linker mutants (0.01 µg, 0.10 µg, and 1.0 µg) indicated by the thickness of the gray wedge at the bottom of the figure in the presence of gH (0.5 µg), gL (0.5 µg), gB (0.5 µg), and T7 polymerase (0.8 µg). Twenty-four hours posttransfection, cells were detached and overlaid with Daudi B cells expressing HLA class II and T7 promoter. Zero, 2 or 20 µl of sgp42 supernatant was added at the time of overlay (white, gray, and black bars). Twenty hours after the overlay, fusion activity was assessed by the addition of passive lysis buffer, followed by the addition of luciferase substrate. Luciferase activity was measured with a PerkinElmer Victor plate reader. Data shown are results representative of the results of three independent experiments. Error bars represent standard deviations for the normalized values.

    Article Snippet: The medium was changed 16 h posttransfection, and 1 h later, the cells were detached with Versene, counted using a Beckman Coulter Z1 particle counter, 37,500 cells were transferred to each well of a 96-well plate, and the total volume was adjusted to 150 µl with complete Ham’s F-12 medium.

    Techniques: Activated Clotting Time Assay, Dominant Negative Mutation, Transfection, Expressing, Activity Assay, Lysis, Luciferase

    All of the gp42 linker mutants are expressed on the cell surface and are cleaved and secreted. CHO-KI cells were transiently transfected in Opti-MEM medium (Gibco) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected overnight with 4 µg each vector alone (pCAGGS), wild-type gp42, d37-41gp42, d36FLAGgp42, d37-41(I27-1), d37-41(I27-1,2), and d31-44 (I27-1,2,3,4), followed by a medium change to Ham’s F-12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Forty-eight hours posttransfection, the supernatants were collected, and the cells were lysed in Triton X-100 lysis buffer containing protease inhibitors. (A and B) Cell lysates (A) and culture supernatants (B) were analyzed by 12% SDS-PAGE and Western blotting with polyclonal anti-gp42 antibody (PB114; R. Longnecker) and secondary IRDye 800CW-conjugated goat (polyclonal) anti-rabbit IgG (H+L) (catalog no. 926-32211; Li-Cor Biosciences) as previously described. Blots were washed and analyzed with Li-Cor Biosciences Odyssey infrared imaging studio software. The positions of molecular size markers (in kilodaltons) are indicated to the left of the blots. Although the blots in panels A and B were from the same gel, intervening lanes that were not applicable to the experiment shown were removed.

    Journal: mBio

    Article Title: Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

    doi: 10.1128/mBio.02285-14

    Figure Lengend Snippet: All of the gp42 linker mutants are expressed on the cell surface and are cleaved and secreted. CHO-KI cells were transiently transfected in Opti-MEM medium (Gibco) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected overnight with 4 µg each vector alone (pCAGGS), wild-type gp42, d37-41gp42, d36FLAGgp42, d37-41(I27-1), d37-41(I27-1,2), and d31-44 (I27-1,2,3,4), followed by a medium change to Ham’s F-12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Forty-eight hours posttransfection, the supernatants were collected, and the cells were lysed in Triton X-100 lysis buffer containing protease inhibitors. (A and B) Cell lysates (A) and culture supernatants (B) were analyzed by 12% SDS-PAGE and Western blotting with polyclonal anti-gp42 antibody (PB114; R. Longnecker) and secondary IRDye 800CW-conjugated goat (polyclonal) anti-rabbit IgG (H+L) (catalog no. 926-32211; Li-Cor Biosciences) as previously described. Blots were washed and analyzed with Li-Cor Biosciences Odyssey infrared imaging studio software. The positions of molecular size markers (in kilodaltons) are indicated to the left of the blots. Although the blots in panels A and B were from the same gel, intervening lanes that were not applicable to the experiment shown were removed.

    Article Snippet: The medium was changed 16 h posttransfection, and 1 h later, the cells were detached with Versene, counted using a Beckman Coulter Z1 particle counter, 37,500 cells were transferred to each well of a 96-well plate, and the total volume was adjusted to 150 µl with complete Ham’s F-12 medium.

    Techniques: Transfection, Plasmid Preparation, Lysis, SDS Page, Western Blot, Imaging, Software

    Mapping of the activation domain of Luman by deletion analysis. On the left is a schematic representation of the structure of the Luman protein, in which numbers indicate the positions of the amino acids. The X in the HCF-binding domain of D78A represents a point mutation (alanine replaces the aspartate residue at position 78). Features of the protein discussed in the text are labeled. Luman and its deletion mutants were fused to the GAL4 DBD. The same amount (0.5 μg) of each plasmid was introduced into COS7 cells along with the reporter plasmid, pG5EC (0.5 μg), which has five copies of GAL4 UAS in the promoter region linked to the cat gene. CAT activity was measured by ELISA 48 h posttransfection.

    Journal: Journal of Virology

    Article Title: The Herpesvirus Transactivator VP16 Mimics a Human Basic Domain Leucine Zipper Protein, Luman, in Its Interaction with HCF

    doi:

    Figure Lengend Snippet: Mapping of the activation domain of Luman by deletion analysis. On the left is a schematic representation of the structure of the Luman protein, in which numbers indicate the positions of the amino acids. The X in the HCF-binding domain of D78A represents a point mutation (alanine replaces the aspartate residue at position 78). Features of the protein discussed in the text are labeled. Luman and its deletion mutants were fused to the GAL4 DBD. The same amount (0.5 μg) of each plasmid was introduced into COS7 cells along with the reporter plasmid, pG5EC (0.5 μg), which has five copies of GAL4 UAS in the promoter region linked to the cat gene. CAT activity was measured by ELISA 48 h posttransfection.

    Article Snippet: CAT activity was measured 48 h posttransfection, using the CAT ELISA kit (Boehringer Mannheim).

    Techniques: Activation Assay, Binding Assay, Mutagenesis, Labeling, Plasmid Preparation, Activity Assay, Enzyme-linked Immunosorbent Assay

    Morphology of PA-457-resistant virus particles. Thin-section transmission EM analysis of virions with the indicated mutations produced from HeLa cells cultured without PA-457 or with 5.0 μg/ml PA-457 is shown. Cells were fixed at 48 h posttransfection and were analyzed by EM. (a to c) WT pNL4-3 without (a) or with (b and c) PA-457; (d and e) SP1-A1V without (d) or with (e) PA-457; (f to i) SP1-A3V without PA-457; (j to l) SP1-A3T without PA-457; (m and n) SP1-A3V with PA-457; (o to q) SP1-A3T with PA-457; (r and s) CA-G225S/SP1-A3V without (r) or with (s) PA-457. Bars, 200 nm.

    Journal: Journal of Virology

    Article Title: In Vitro Resistance to the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PA-457 (Bevirimat) ▿

    doi: 10.1128/JVI.01369-06

    Figure Lengend Snippet: Morphology of PA-457-resistant virus particles. Thin-section transmission EM analysis of virions with the indicated mutations produced from HeLa cells cultured without PA-457 or with 5.0 μg/ml PA-457 is shown. Cells were fixed at 48 h posttransfection and were analyzed by EM. (a to c) WT pNL4-3 without (a) or with (b and c) PA-457; (d and e) SP1-A1V without (d) or with (e) PA-457; (f to i) SP1-A3V without PA-457; (j to l) SP1-A3T without PA-457; (m and n) SP1-A3V with PA-457; (o to q) SP1-A3T with PA-457; (r and s) CA-G225S/SP1-A3V without (r) or with (s) PA-457. Bars, 200 nm.

    Article Snippet: Twenty-four hours posttransfection, cells were starved for 30 min at 37°C in Cys/Met-free medium and pulse-labeled in the same medium for 15 min at 37°C using 50 μCi of [35 S]Cys/Met Pro-mix (Amersham).

    Techniques: Transmission Assay, Produced, Cell Culture

    Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h posttransfection. Six pictures per condition were analyzed with the CellProfiler program to determine percentages of positive cells or mean integrated intensities of stained cells. **, P

    Journal: Journal of Virology

    Article Title: Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

    doi: 10.1128/JVI.01198-17

    Figure Lengend Snippet: Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h posttransfection. Six pictures per condition were analyzed with the CellProfiler program to determine percentages of positive cells or mean integrated intensities of stained cells. **, P

    Article Snippet: Sixteen hours posttransfection, cells were treated with MG-132 (final concentration, 2 μM; Santa Cruz Biotechnology).

    Techniques: Microscopy, Staining

    Intracellular localization of AAP proteins of 10 different AAV serotypes and correlation with VP expression and capsid formation. (A) Expression plasmids encoding AAPs of the AAV serotypes shown were transfected together with a corresponding AAP-deficient helper plasmid into HeLa cells. AAP was detected by staining with an anti-HA tag antibody at 42 h posttransfection. Nuclei were stained with Hoechst stain. Transfections and immunostainings were performed in LabTek format. Images were captured with a Leica TCS SP5 microscope. Scale bar, 20 μm. (B and C) HEK293T cells were cotransfected in LabTek slides with AAP-deficient helpers of the serotypes shown, together with expression plasmids encoding cognate HA-tagged AAP, a luciferase vector, and an adenoviral helper. Free VP proteins (B) or assembled capsids (C) were detected using B1/VP or the A20/ADK antibodies, respectively. AAP was stained with an anti-HA tag antibody, and nuclei were stained with Hoechst stain. Pictures (not shown) were taken with an automated Olympus microscope at 20 h or 42 h posttransfection. Shown are normalized cross correlation coefficients (NC) that were determined as a measure of colocalization of the red (HA) and green (VP [B] or capsids [C]) color channel. An NC value of −1 indicates complete disagreement, and +1 indicates complete overlap of the two stains.

    Journal: Journal of Virology

    Article Title: Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

    doi: 10.1128/JVI.01198-17

    Figure Lengend Snippet: Intracellular localization of AAP proteins of 10 different AAV serotypes and correlation with VP expression and capsid formation. (A) Expression plasmids encoding AAPs of the AAV serotypes shown were transfected together with a corresponding AAP-deficient helper plasmid into HeLa cells. AAP was detected by staining with an anti-HA tag antibody at 42 h posttransfection. Nuclei were stained with Hoechst stain. Transfections and immunostainings were performed in LabTek format. Images were captured with a Leica TCS SP5 microscope. Scale bar, 20 μm. (B and C) HEK293T cells were cotransfected in LabTek slides with AAP-deficient helpers of the serotypes shown, together with expression plasmids encoding cognate HA-tagged AAP, a luciferase vector, and an adenoviral helper. Free VP proteins (B) or assembled capsids (C) were detected using B1/VP or the A20/ADK antibodies, respectively. AAP was stained with an anti-HA tag antibody, and nuclei were stained with Hoechst stain. Pictures (not shown) were taken with an automated Olympus microscope at 20 h or 42 h posttransfection. Shown are normalized cross correlation coefficients (NC) that were determined as a measure of colocalization of the red (HA) and green (VP [B] or capsids [C]) color channel. An NC value of −1 indicates complete disagreement, and +1 indicates complete overlap of the two stains.

    Article Snippet: Sixteen hours posttransfection, cells were treated with MG-132 (final concentration, 2 μM; Santa Cruz Biotechnology).

    Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Microscopy, Luciferase

    Evidence for proteasomal degradation of free VP proteins in the absence of AAP. (A) Workflow for AAV crude cell lysate production with MG-132 treatment at 5 and/or 24 h posttransfection. AAV, AAV2 helper plasmid expressing rep and cap ; Ad, adenoviral helper plasmid; GFP, GFP-encoding AAV vector plasmid. (B) Western blot analysis of cell lysates obtained from the workflow in panel A. Cells were either left untreated (left blot) or incubated with MG-132 at 5 h (middle blot) and/or 24 h (right blot) after transfection. VP1 to VP3 proteins were detected with the B1 antibody. (C) Quantification of relative GFP expression in HEK293T cells at 48 h after transduction with AAV crude cell lysates (in principle, the same samples as for panel B). Positive cells were counted via flow cytometry and values normalized to the AAV2wt control without AAP2 (set to 1.0). Shown are means from three independent biological replicates plus SD. (D and E) Effect of proteasomal inhibition on AAP/VP/capsid localization. HEK293T cells were transfected with APP2mut (D) or cotransfected with AAP2mut and HA-tagged AAP2 (E) prior to staining with antibodies against free VP proteins (B1, green, upper panels) or assembled capsids (A20, green, lower panels) or DAPI (nuclei). The images illustrate the localization of AAP2 (red) and VP/capsid (B1 or A20, green) in the presence or absence of MG-132. Overlay images are shown on the right side of each panel. Arrowheads highlight examples of clear VP/capsid (B1/A20) and AAP2 colocalization in the nuclei of transfected cells. Insets show a higher magnification of selected areas. The images shown are maximum projections of deconvolved confocal optical sections spanning the entire volume of cells. All images are representative of three independent experiments. Scale bar, 5 μm; scale bar in insets, 2 μm.

    Journal: Journal of Virology

    Article Title: Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

    doi: 10.1128/JVI.01198-17

    Figure Lengend Snippet: Evidence for proteasomal degradation of free VP proteins in the absence of AAP. (A) Workflow for AAV crude cell lysate production with MG-132 treatment at 5 and/or 24 h posttransfection. AAV, AAV2 helper plasmid expressing rep and cap ; Ad, adenoviral helper plasmid; GFP, GFP-encoding AAV vector plasmid. (B) Western blot analysis of cell lysates obtained from the workflow in panel A. Cells were either left untreated (left blot) or incubated with MG-132 at 5 h (middle blot) and/or 24 h (right blot) after transfection. VP1 to VP3 proteins were detected with the B1 antibody. (C) Quantification of relative GFP expression in HEK293T cells at 48 h after transduction with AAV crude cell lysates (in principle, the same samples as for panel B). Positive cells were counted via flow cytometry and values normalized to the AAV2wt control without AAP2 (set to 1.0). Shown are means from three independent biological replicates plus SD. (D and E) Effect of proteasomal inhibition on AAP/VP/capsid localization. HEK293T cells were transfected with APP2mut (D) or cotransfected with AAP2mut and HA-tagged AAP2 (E) prior to staining with antibodies against free VP proteins (B1, green, upper panels) or assembled capsids (A20, green, lower panels) or DAPI (nuclei). The images illustrate the localization of AAP2 (red) and VP/capsid (B1 or A20, green) in the presence or absence of MG-132. Overlay images are shown on the right side of each panel. Arrowheads highlight examples of clear VP/capsid (B1/A20) and AAP2 colocalization in the nuclei of transfected cells. Insets show a higher magnification of selected areas. The images shown are maximum projections of deconvolved confocal optical sections spanning the entire volume of cells. All images are representative of three independent experiments. Scale bar, 5 μm; scale bar in insets, 2 μm.

    Article Snippet: Sixteen hours posttransfection, cells were treated with MG-132 (final concentration, 2 μM; Santa Cruz Biotechnology).

    Techniques: Plasmid Preparation, Expressing, Western Blot, Incubation, Transfection, Transduction, Flow Cytometry, Cytometry, Inhibition, Staining

    Proof that AAP fosters assembly of capsids of multiple AAV serotypes. (A) Capsids of the eight AAV serotypes shown were produced by cotransfecting HEK293T cells with the corresponding wild-type or AAP-deficient (mutant) AAV helper, an AAV vector, and an adenoviral helper. Following freeze-thaw lysis at 3 days posttransfection, capsid-containing supernatants were spotted onto nitrocellulose membranes under nondenaturing conditions. In every other lane, the capsids were heat denatured via incubation at 95°C for 5 min. Intact capsids were detected with the ADK series of antibodies (ADK8 cross-reacts with AAV3) or A20 (for AAV2). For most of the shown AAV serotypes (AAV2, -3, -6, -8, and -9), lack of AAP prevented formation of assembled capsids. Arrows highlight exceptions, i.e., signals detected with the ADK antibodies for AAV1, -4, and -5 even in the absence of AAP. Note that low or no signals were detected with the VP serum or B1 antibody following heat denaturation of the same samples (arrowhead). The blots shown are representative of three independent experiments, which all gave similar results. (B) Further investigation of the three exceptions from panel A, i.e., AAV serotypes 1, 4, and 5. In lanes S, the samples were not only heated but additionally treated with SDS (see Materials and Methods for details). The arrowhead highlights the particular resistance of the AAP-independent AAV5 structures even to these harsh denaturing conditions. (C) Stabilization of free VP proteins by MG-132 treatment does not enhance capsid assembly. Capsid were produced, blotted, and detected as described for panel A. Instead of heat and/or SDS denaturation, samples in lane MG were derived from cells that were treated with the proteasome inhibitor MG-132 during vector production. At 5 and 24 h after transfection, the medium was changed to fresh medium containing MG-132, and 48 h later, supernatants from the lysed cells were analyzed. Note that AAV4 and AAV5, as well as, to a lesser extent, AAV1 (and AAV6), gave signals even in the absence of AAP, independently confirming the data in panels A and B. The blots shown are representative of two independent experiments, which gave similar results.

    Journal: Journal of Virology

    Article Title: Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

    doi: 10.1128/JVI.01198-17

    Figure Lengend Snippet: Proof that AAP fosters assembly of capsids of multiple AAV serotypes. (A) Capsids of the eight AAV serotypes shown were produced by cotransfecting HEK293T cells with the corresponding wild-type or AAP-deficient (mutant) AAV helper, an AAV vector, and an adenoviral helper. Following freeze-thaw lysis at 3 days posttransfection, capsid-containing supernatants were spotted onto nitrocellulose membranes under nondenaturing conditions. In every other lane, the capsids were heat denatured via incubation at 95°C for 5 min. Intact capsids were detected with the ADK series of antibodies (ADK8 cross-reacts with AAV3) or A20 (for AAV2). For most of the shown AAV serotypes (AAV2, -3, -6, -8, and -9), lack of AAP prevented formation of assembled capsids. Arrows highlight exceptions, i.e., signals detected with the ADK antibodies for AAV1, -4, and -5 even in the absence of AAP. Note that low or no signals were detected with the VP serum or B1 antibody following heat denaturation of the same samples (arrowhead). The blots shown are representative of three independent experiments, which all gave similar results. (B) Further investigation of the three exceptions from panel A, i.e., AAV serotypes 1, 4, and 5. In lanes S, the samples were not only heated but additionally treated with SDS (see Materials and Methods for details). The arrowhead highlights the particular resistance of the AAP-independent AAV5 structures even to these harsh denaturing conditions. (C) Stabilization of free VP proteins by MG-132 treatment does not enhance capsid assembly. Capsid were produced, blotted, and detected as described for panel A. Instead of heat and/or SDS denaturation, samples in lane MG were derived from cells that were treated with the proteasome inhibitor MG-132 during vector production. At 5 and 24 h after transfection, the medium was changed to fresh medium containing MG-132, and 48 h later, supernatants from the lysed cells were analyzed. Note that AAV4 and AAV5, as well as, to a lesser extent, AAV1 (and AAV6), gave signals even in the absence of AAP, independently confirming the data in panels A and B. The blots shown are representative of two independent experiments, which gave similar results.

    Article Snippet: Sixteen hours posttransfection, cells were treated with MG-132 (final concentration, 2 μM; Santa Cruz Biotechnology).

    Techniques: Produced, Mutagenesis, Plasmid Preparation, Lysis, Incubation, Derivative Assay, Transfection

    IL-6 activates Etk through PI3-kinase. ( A ) Wortmannin (Wort) blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk. At 24 hr posttransfection, the cells were serum starved for 24 hr. The cells were then pretreated with 100 nM wortmannin (+) or dimethyl sulfoxide Mock (−) for 30 min followed by IL-6 treatment for 30 min. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-T7 antibody ( Lower ). The tyrosine phosphorylation of the T7-tagged Etk is indicated. ( B ) A myrislated p110 activates Etk. The CV-1 cells were transfected by pcDNA3-T7-Etk in the presence (+) or absence (−) of pcDNA3-p110 myr. At 24 hr posttransfection, the cells were serum starved for 24 hr. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with anti-phosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ). ( C ) A dominant negative p85 blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk (wild type) or pcDNA3-T7-EtkΔPH(deletion of PH domain) in the presence (+) or absence (−) of pcDNA3-p85DN. At 24 hr posttransfection, the cells were serum starved for 24 hr followed by IL-6 treatment for 30 min. The cell extracts from each treatment were subjected to immunoprecipitation by anti-T7 antibody, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ).

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

    Article Title: Etk/Bmx, a tyrosine kinase with a pleckstrin-homology domain, is an effector of phosphatidylinositol 3?-kinase and is involved in interleukin 6-induced neuroendocrine differentiation of prostate cancer cells

    doi:

    Figure Lengend Snippet: IL-6 activates Etk through PI3-kinase. ( A ) Wortmannin (Wort) blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk. At 24 hr posttransfection, the cells were serum starved for 24 hr. The cells were then pretreated with 100 nM wortmannin (+) or dimethyl sulfoxide Mock (−) for 30 min followed by IL-6 treatment for 30 min. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-T7 antibody ( Lower ). The tyrosine phosphorylation of the T7-tagged Etk is indicated. ( B ) A myrislated p110 activates Etk. The CV-1 cells were transfected by pcDNA3-T7-Etk in the presence (+) or absence (−) of pcDNA3-p110 myr. At 24 hr posttransfection, the cells were serum starved for 24 hr. The cell extracts were subjected to immunoprecipitation by anti-T7 antibody respectively, followed by Western blot analysis with anti-phosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ). ( C ) A dominant negative p85 blocks the activation of Etk by IL-6. The CV-1 cells were transfected with pcDNA3-T7-Etk (wild type) or pcDNA3-T7-EtkΔPH(deletion of PH domain) in the presence (+) or absence (−) of pcDNA3-p85DN. At 24 hr posttransfection, the cells were serum starved for 24 hr followed by IL-6 treatment for 30 min. The cell extracts from each treatment were subjected to immunoprecipitation by anti-T7 antibody, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ).

    Article Snippet: At 24hrs posttransfection, the cells were serum starved for 24 hr followed by treatment with IL-6 (Upstate Biotechnology, Lake Placid, NY) as indicated in the figure legends.

    Techniques: Activation Assay, Transfection, Immunoprecipitation, Western Blot, Dominant Negative Mutation

    Modulation of Etk kinase activity by point mutations in PH and kinase domains. ( A ) Substitution of glutamate 42 of Etk with lysine diminishes the Etk activity in vivo . The CV-1 cells were transfected with pcDNA3-T7-Etk (wild type), pcDNA3-T7-EtkE42K or pcDNA3-T7-EtkDN (E42K+ K444Q). At 48 hr posttransfection, the cells were lysed and cell extracts from each treatment were subjected to immunoprecipitation by anti-T7 antibody, respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ). ( B ) In vitro kinase assays of the immunoprecipitates from A reveals diminished kinase activity of EtkE42K and abolished activity of EtkDN. ( C ) EtkDN is a dominant negative mutant. The CV-1 cells were transfected with the plasmids indicated. The ratio between the HA-tagged Etk plasmids and T7-tagged Etk plasmids or vector (pcDNA3) used in the transfections is 1:4. At 48 hr posttransfection, the cells were lysed and cell extracts were subjected to immunoprecipitation by anti-HA antibody respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-HA antibody ( Lower ).

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

    Article Title: Etk/Bmx, a tyrosine kinase with a pleckstrin-homology domain, is an effector of phosphatidylinositol 3?-kinase and is involved in interleukin 6-induced neuroendocrine differentiation of prostate cancer cells

    doi:

    Figure Lengend Snippet: Modulation of Etk kinase activity by point mutations in PH and kinase domains. ( A ) Substitution of glutamate 42 of Etk with lysine diminishes the Etk activity in vivo . The CV-1 cells were transfected with pcDNA3-T7-Etk (wild type), pcDNA3-T7-EtkE42K or pcDNA3-T7-EtkDN (E42K+ K444Q). At 48 hr posttransfection, the cells were lysed and cell extracts from each treatment were subjected to immunoprecipitation by anti-T7 antibody, respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-Etk antibody ( Lower ). ( B ) In vitro kinase assays of the immunoprecipitates from A reveals diminished kinase activity of EtkE42K and abolished activity of EtkDN. ( C ) EtkDN is a dominant negative mutant. The CV-1 cells were transfected with the plasmids indicated. The ratio between the HA-tagged Etk plasmids and T7-tagged Etk plasmids or vector (pcDNA3) used in the transfections is 1:4. At 48 hr posttransfection, the cells were lysed and cell extracts were subjected to immunoprecipitation by anti-HA antibody respectively, followed by Western blot analysis with antiphosphotyrosine antibody ( Upper ) and anti-HA antibody ( Lower ).

    Article Snippet: At 24hrs posttransfection, the cells were serum starved for 24 hr followed by treatment with IL-6 (Upstate Biotechnology, Lake Placid, NY) as indicated in the figure legends.

    Techniques: Activity Assay, In Vivo, Transfection, Immunoprecipitation, Western Blot, In Vitro, Dominant Negative Mutation, Plasmid Preparation

    NRIP blocks the polyubiquitination and phosphorylation of HPV-16 E2 through its CaM binding domain. (A) The expression vectors for HA-tagged ubiquitin, EGFP-16E2, NRIP-FLAG, and NRIPΔIQ-FLAG were transfected into 293 cells. At 40 h posttransfection,

    Journal: Journal of Virology

    Article Title: NRIP, a Novel Calmodulin Binding Protein, Activates Calcineurin To Dephosphorylate Human Papillomavirus E2 Protein ▿

    doi: 10.1128/JVI.02453-10

    Figure Lengend Snippet: NRIP blocks the polyubiquitination and phosphorylation of HPV-16 E2 through its CaM binding domain. (A) The expression vectors for HA-tagged ubiquitin, EGFP-16E2, NRIP-FLAG, and NRIPΔIQ-FLAG were transfected into 293 cells. At 40 h posttransfection,

    Article Snippet: At 36 h posttransfection, cells were treated with cycloheximide to block de novo protein synthesis.

    Techniques: Chick Chorioallantoic Membrane Assay, Binding Assay, Expressing, Transfection