e1b Search Results


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  • 85
    Millipore ad5 e1b
    Selective expression of E1A in MUC1-positive cells infected with Ad.DF3-E1. ( a ) The indicated cells were incubated with either mAb DF3 (open area) or an isotype-identical control Ab (shaded area) and then subjected to flow cytometric analysis. ( b ) Cells were infected with Ad.DF3-E1, Ad.CMV–β-gal, or wild-type <t>Ad5.</t> Lysates from control (noninfected) and infected cells were subjected to immunoblot analysis with anti-E1A Ab. ( c ) Lysates from Ad.DF3-E1–infected cells were subjected to immunoblot analysis with <t>anti-E1B.</t>
    Ad5 E1b, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e1b 55k
    <t>E1B-55K/E4orf3</t> double mutant virus-infected cells express reduced levels of late viral proteins and increased levels of cellular proteins. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 20 PFU per cell. The status of the relevant viral genes is indicated below the gel. At 36 h postinfection, cells were labeled with 35 S-labeled amino acids for 1 h. Proteins from 10 5 cells (per lane) were separated by SDS-PAGE. Radioactive proteins were visualized by phosphorescence imaging. The approximate migration and mass (in kilodaltons) of molecular size standards are indicated to the right of the gel. The positions of prominent viral and cellular proteins were determined with adenovirus virion standards labeled with [ 14 C]amino acids or by immunoprecipitation and are indicated to the left of the image. The gel shown is representative of three independent experiments.
    E1b 55k, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Biomatik e1b tol2
    In vivo regulatory screen of all 6-mers . (a) Left top: naively concatenating three 6-mers creates an oligomer with multiple representatives of ATTGCG (red bars). Center and left: cartoon of a de Bruijn graph. Nodes (colored boxes) represent 6-mers; edges (arrows) represent overlap. A standard de-Bruijn-sequence library is built from one path that traverses each of 4,096 nodes once. Constructed from multiple paths, our MRCC library instead uses one representative for each pair of reverse-complementary 6-mers (green and yellow; self-reverse-complementary palindromes in blue), making it nearly 50% smaller (Additional file 1 ). Right: 16 of 16,384 edges shown. Our algorithm removes reverse-complementary paths (black, red) between palindrome pairs and then decomposes the remaining graph into reverse-complementary cycles. It allowed us to design an ultra-compact library of DNA sequences with uniform 6-mer coverage. (b) Schematic depicting the sub-cloning of each 15-bp multiplexed oligomer into the <t>E1b-Tol2</t> vector and subsequent injection into single-cell zebrafish embryos. (c) Violin plots depicting the distribution of the expression patterns of each tissue at 24 hpf. White lines indicate the fractional expression values for the empty vector construct. (d) Scatter plot depicting the method by which we selected consistently expressed multiplexed oligomers whose expression was not significantly correlated to minimal-promoter bias. The vertical dotted line denotes the 40% fractional expression threshold that was used, whereas the horizontal dotted line corresponds to a false discovery rate-adjusted P -value of 0.05. (e) Histogram depicting the tissue specificity of the 22 uncorrelated, consistently expressed constructs at 24 hpf. (f) Representative images at 24 hpf for embryos injected with 1EF03 (top) and 3EF09 (bottom), exhibiting broad expression that was correlated with that of the empty vector expression. The full sequence of each construct (with XhoI and BglII flanking sites) is listed below each figure. Both constructs have a 5' GC-rich region. GFP, green fluorescent protein.
    E1b Tol2, supplied by Biomatik, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Proteintech anti e1b ap5
    The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, <t>E1B-AP5,</t> and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .
    Anti E1b Ap5, supplied by Proteintech, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    ZIRC Inc uas e1b
    Phenotypic characterization of a stable transgenic line. ( a ) Graphic representation of the DNA constructs used for transgenesis and breeding. ( b ) A 24 h.p.f. embryo expresses Smoa1-EGFP in the forebrain and retinal neuroblasts. A 96 h.p.f. double transgenic embryo derived from crossing of the Tg(krt5:Gal4VP16;14 × <t>UAS:smoa1-EGFP)</t> and the <t>Tg(UAS-E1b:nfsB-mCherry)</t> c264 stable lines expressed GFP ( c ) and mCherry ( d ) in the ONs (arrows). ( e ) A 6-month-old wild-type and ( f ) a transgenic fish with a gross tumor in its left eye (arrow). ( g ) Wild-type fish formed a normal optic chiasm, whereas ( h ) transgenic fish failed to form optic chiasm and showed dramatic expansion of the ONH region (arrow). ( i ) Dissected brain and eyes showing an enlarged ON associating with a gross eye tumor (arrow).
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    91
    becton dickinson e1b 19k
    Phenotypic characterization of a stable transgenic line. ( a ) Graphic representation of the DNA constructs used for transgenesis and breeding. ( b ) A 24 h.p.f. embryo expresses Smoa1-EGFP in the forebrain and retinal neuroblasts. A 96 h.p.f. double transgenic embryo derived from crossing of the Tg(krt5:Gal4VP16;14 × <t>UAS:smoa1-EGFP)</t> and the <t>Tg(UAS-E1b:nfsB-mCherry)</t> c264 stable lines expressed GFP ( c ) and mCherry ( d ) in the ONs (arrows). ( e ) A 6-month-old wild-type and ( f ) a transgenic fish with a gross tumor in its left eye (arrow). ( g ) Wild-type fish formed a normal optic chiasm, whereas ( h ) transgenic fish failed to form optic chiasm and showed dramatic expansion of the ONH region (arrow). ( i ) Dissected brain and eyes showing an enlarged ON associating with a gross eye tumor (arrow).
    E1b 19k, supplied by becton dickinson, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Proteintech e1b ap5
    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, <t>EIB-AP5</t> or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using <t>E1B-AP5</t> antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.
    E1b Ap5, supplied by Proteintech, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e1b 19kda specific antibody
    Construction and characterization of viruses . (A) Transcriptional activity of the AFP promoter in different cell lines. The AFP promoter with an SV40 enhancer was incorporated into pGL3-basic. The relative activity of pGL3-SV40EAFP to pGL3-Control in various cell lines are presented as the mean+SD (n = 3). (B) Schematic structure of the viruses Ad.SPDD, Ad.SPDD-HCCS1, and Ad.SPDD-HCCS1. ITR, inverted terminal repeat; ψ, viral packing signal. Ad.SPDD was quadruply modified compared to the wild-type adenovirus; the modifications were the deletion of E1B19K, the deletion of E1B55K, and the deletion of 24 bp of E1A, as well as deletion of the survivin promoter controlling E1A expression. HCCS1 or HA-tagged HCCS1 in a cassette was carried by Ad.SPDD. (C) Characterization of viruses by western blot analysis. Huh-7 cells were infected with the indicated viruses, and the levels of the expressed E1A, <t>E1B</t> <t>19KDa,</t> E1B 55KDa and HA-Tag were analyzed 48 hours post infection (h.p.i), with actin as loading control. (***: P
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    Onyx Pharmaceuticals e1b deletion
    Construction and characterization of viruses . (A) Transcriptional activity of the AFP promoter in different cell lines. The AFP promoter with an SV40 enhancer was incorporated into pGL3-basic. The relative activity of pGL3-SV40EAFP to pGL3-Control in various cell lines are presented as the mean+SD (n = 3). (B) Schematic structure of the viruses Ad.SPDD, Ad.SPDD-HCCS1, and Ad.SPDD-HCCS1. ITR, inverted terminal repeat; ψ, viral packing signal. Ad.SPDD was quadruply modified compared to the wild-type adenovirus; the modifications were the deletion of E1B19K, the deletion of E1B55K, and the deletion of 24 bp of E1A, as well as deletion of the survivin promoter controlling E1A expression. HCCS1 or HA-tagged HCCS1 in a cassette was carried by Ad.SPDD. (C) Characterization of viruses by western blot analysis. Huh-7 cells were infected with the indicated viruses, and the levels of the expressed E1A, <t>E1B</t> <t>19KDa,</t> E1B 55KDa and HA-Tag were analyzed 48 hours post infection (h.p.i), with actin as loading control. (***: P
    E1b Deletion, supplied by Onyx Pharmaceuticals, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher e1b ap5
    (A) Localization of <t>E1B-AP5,</t> ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.
    E1b Ap5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e1b 19k
    (A) Localization of <t>E1B-AP5,</t> ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.
    E1b 19k, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech e1b ap5 polyclonal antibody
    (A) Localization of <t>E1B-AP5,</t> ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.
    E1b Ap5 Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti e1b 21kd antibody
    (A) Localization of <t>E1B-AP5,</t> ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.
    Anti E1b 21kd Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc ac ds e1b egfp vector
    (A) Localization of <t>E1B-AP5,</t> ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.
    Ac Ds E1b Egfp Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    TaKaRa adenovirus e1b
    (A) Localization of <t>E1B-AP5,</t> ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.
    Adenovirus E1b, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Genewiz e1b gene
    Clusters of coregulated cellular RNAs 30 h after infection with Ad5 or <t>E1B</t> mutants. The expression profiles of significantly regulated genes were clustered into eight distinct groups with similar expression response profiles across the different Ad5 genotypes as described in Materials and Methods. Each cluster was searched for statistically significant enrichment of genes that code for proteins with common functions ( P values are Bonferroni corrected for multiple hypothesis testing). GO terms enriched in clusters 2, 3, and 4 are shown at right, with the corresponding P values. Genes that increased and decreased in expression are shown in yellow and blue, respectively, as indicated on the scale bar. hpi, hours postinfection.
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    85
    Millipore hav5 e1b 21kda
    Immunoprecipitation of <t>HAV5</t> E1 proteins from extracts of BHH cell lines with HAV5 E1-specific antibodies. A549, A549-infected with HAV5, 293-Puro, BHH2C, BHH3, and BHH8 cells were labeled with Trans[ 35 S]-label, and cell extracts were immunoprecipitated with anti-HAV2 E1A (13S-5; Santa Cruz) (A), anti-HAV2 <t>E1B-21kDa</t> (3D11; Calbiochem) (B), and anti-HAV5 E1B-55kDa (9C10; Calbiochem) (C) antibodies. Each HAV5 E1 protein is indicated by an arrow. The position of the molecular weight marker is shown on the right side of the panel.
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    Oncogene Science Inc e1b 19k rat mab
    Immunoprecipitation of <t>HAV5</t> E1 proteins from extracts of BHH cell lines with HAV5 E1-specific antibodies. A549, A549-infected with HAV5, 293-Puro, BHH2C, BHH3, and BHH8 cells were labeled with Trans[ 35 S]-label, and cell extracts were immunoprecipitated with anti-HAV2 E1A (13S-5; Santa Cruz) (A), anti-HAV2 <t>E1B-21kDa</t> (3D11; Calbiochem) (B), and anti-HAV5 E1B-55kDa (9C10; Calbiochem) (C) antibodies. Each HAV5 E1 protein is indicated by an arrow. The position of the molecular weight marker is shown on the right side of the panel.
    E1b 19k Rat Mab, supplied by Oncogene Science Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ad14 e1b 20k
    Reduced <t>E1B</t> <t>20K</t> expression correlated with loss of Ad14p1 CPE corpse repression of NF-κB activation. (A) Immunoblot analysis of E1B 19/20K expression in A549 cells infected with wt <t>Ad14,</t> wt Ad5, or Ad14p1 clinical isolate 1986T. (B) RT-qPCR analysis
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    Genewiz e1b mutation
    Construction and characterization of the <t>E1B</t> 55-kDa- and E4 Orf3-null double-mutant virus AdEasyE1Δ2347-dl341. (A) The origins of the segments of the double mutant virus genome are shown schematically, indicating the positions of the single-pair
    E1b Mutation, supplied by Genewiz, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene minimal e1b promoter luciferase gene
    Construction and characterization of the <t>E1B</t> 55-kDa- and E4 Orf3-null double-mutant virus AdEasyE1Δ2347-dl341. (A) The origins of the segments of the double mutant virus genome are shown schematically, indicating the positions of the single-pair
    Minimal E1b Promoter Luciferase Gene, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Oncogene Science Inc e1b 19 kda
    HCMV IE1 and IE2 genes and their products are not present in transformed BRK cell lines. ( A ) Lack of expression of the IE1 or IE2 protein in transformed BRK cell lines when assayed by immunoprecipitation of 35 S-labeled cell extracts using monoclonal antibody mAB810. A-1 is a cell line established by transfection with the E1A-expressing plasmid alone. A/1/2–1 to A/1/2–10 are cell lines transformed by E1A plus IE1 and IE2. Lanes designated HCMV INF display immunoprecipitates from HCMV-infected human foreskin fibroblasts; lane 13 is an exposure 1/8 as long as that presented for lanes 1–12. Numbers to the left of the gel mark the positions of marker proteins whose sizes are indicated in <t>kDa.</t> ( B ) Southern blot analysis showing the lack of IE1 - or IE2 -specific DNA in E1A/IE1/IE2-induced transformants (lanes 1–10). A/B-1 and A/B-2 are cell lines transformed with E1A and <t>E1B-19-kDa.</t> HeLa, 293, and HP1.14 (a derivative HeLa cells expressing IE1) are included as controls. pCGN-IE1 and pCGN-IE2 are IE1 and IE2 expression plasmids, respectively, in amounts equivalent to the rest of the samples, assuming they contain one copy of each of the HCMV genes per cell. Numbers to the left of the gel are marker DNA sizes in kb. ( C ) Expression of E1A protein as determined by immunoprecipitation followed by immunoblot analysis, both with monoclonal antibody M73 to E1A. Lanes are labeled as in A and B . Numbers to the left of the gel are marker protein sizes in kDa. Autoradiograms were scanned and cropped using Photoshop and figures were prepared using Freehand software.
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    Xenobiotics e1b expression modulators
    Effect of overexpression and siRNA knockdown of NRF2 on <t>E1b</t> expression
    E1b Expression Modulators, supplied by Xenobiotics, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals <t>(pG5</t> [reporter plasmid pG5 <t>E1b</t> Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.
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    Image Search Results


    Selective expression of E1A in MUC1-positive cells infected with Ad.DF3-E1. ( a ) The indicated cells were incubated with either mAb DF3 (open area) or an isotype-identical control Ab (shaded area) and then subjected to flow cytometric analysis. ( b ) Cells were infected with Ad.DF3-E1, Ad.CMV–β-gal, or wild-type Ad5. Lysates from control (noninfected) and infected cells were subjected to immunoblot analysis with anti-E1A Ab. ( c ) Lysates from Ad.DF3-E1–infected cells were subjected to immunoblot analysis with anti-E1B.

    Journal: The Journal of Clinical Investigation

    Article Title: Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen

    doi:

    Figure Lengend Snippet: Selective expression of E1A in MUC1-positive cells infected with Ad.DF3-E1. ( a ) The indicated cells were incubated with either mAb DF3 (open area) or an isotype-identical control Ab (shaded area) and then subjected to flow cytometric analysis. ( b ) Cells were infected with Ad.DF3-E1, Ad.CMV–β-gal, or wild-type Ad5. Lysates from control (noninfected) and infected cells were subjected to immunoblot analysis with anti-E1A Ab. ( c ) Lysates from Ad.DF3-E1–infected cells were subjected to immunoblot analysis with anti-E1B.

    Article Snippet: Protein was analyzed by immunoblotting with mAb Ad2 E1A or Ad5 E1B (1 μg/mL; Oncogene Research Products, Cambridge, Massachusetts, USA).

    Techniques: Expressing, Infection, Incubation, Flow Cytometry

    E1B-55K/E4orf3 double mutant virus-infected cells express reduced levels of late viral proteins and increased levels of cellular proteins. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 20 PFU per cell. The status of the relevant viral genes is indicated below the gel. At 36 h postinfection, cells were labeled with 35 S-labeled amino acids for 1 h. Proteins from 10 5 cells (per lane) were separated by SDS-PAGE. Radioactive proteins were visualized by phosphorescence imaging. The approximate migration and mass (in kilodaltons) of molecular size standards are indicated to the right of the gel. The positions of prominent viral and cellular proteins were determined with adenovirus virion standards labeled with [ 14 C]amino acids or by immunoprecipitation and are indicated to the left of the image. The gel shown is representative of three independent experiments.

    Journal: Journal of Virology

    Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background

    doi: 10.1128/JVI.78.18.9924-9935.2004

    Figure Lengend Snippet: E1B-55K/E4orf3 double mutant virus-infected cells express reduced levels of late viral proteins and increased levels of cellular proteins. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 20 PFU per cell. The status of the relevant viral genes is indicated below the gel. At 36 h postinfection, cells were labeled with 35 S-labeled amino acids for 1 h. Proteins from 10 5 cells (per lane) were separated by SDS-PAGE. Radioactive proteins were visualized by phosphorescence imaging. The approximate migration and mass (in kilodaltons) of molecular size standards are indicated to the right of the gel. The positions of prominent viral and cellular proteins were determined with adenovirus virion standards labeled with [ 14 C]amino acids or by immunoprecipitation and are indicated to the left of the image. The gel shown is representative of three independent experiments.

    Article Snippet: Because the cytoplasmic-to-nuclear late mRNA ratio in the double mutant virus-infected cells was the same as in the E1B-55K single mutant virus-infected cells, the loss of E4orf3 function does not appear to exacerbate the defect in viral mRNA transport associated with the loss of E1B-55K.

    Techniques: Mutagenesis, Infection, Labeling, SDS Page, Imaging, Migration, Immunoprecipitation

    (A) Relative levels of cytoplasmic and nuclear L3 and L5 late viral transcripts determined by RNase protection assays. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 20 PFU per cell. At 16 h postinfection, RNA was isolated from both the cytoplasmic and nuclear fractions. L3 and L5 transcript levels were determined by RNase protection. The results of a representative protection assay are shown. (B) E1B-55K/E4orf3 double mutant virus-infected cells contain less late cytoplasmic viral mRNA than cells infected with the wild-type or parent mutant virus. The cytoplasmic levels of L3 and L5 transcripts in HeLa cells infected with the indicated viruses were measured by RNase protection and are expressed relative to the levels in dl 309-infected cells, which were set to 100. The status of the relevant genes is indicated below the histogram. The results are averages obtained from four to six experiments. Error bars indicate the standard error of the mean.

    Journal: Journal of Virology

    Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background

    doi: 10.1128/JVI.78.18.9924-9935.2004

    Figure Lengend Snippet: (A) Relative levels of cytoplasmic and nuclear L3 and L5 late viral transcripts determined by RNase protection assays. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 20 PFU per cell. At 16 h postinfection, RNA was isolated from both the cytoplasmic and nuclear fractions. L3 and L5 transcript levels were determined by RNase protection. The results of a representative protection assay are shown. (B) E1B-55K/E4orf3 double mutant virus-infected cells contain less late cytoplasmic viral mRNA than cells infected with the wild-type or parent mutant virus. The cytoplasmic levels of L3 and L5 transcripts in HeLa cells infected with the indicated viruses were measured by RNase protection and are expressed relative to the levels in dl 309-infected cells, which were set to 100. The status of the relevant genes is indicated below the histogram. The results are averages obtained from four to six experiments. Error bars indicate the standard error of the mean.

    Article Snippet: Because the cytoplasmic-to-nuclear late mRNA ratio in the double mutant virus-infected cells was the same as in the E1B-55K single mutant virus-infected cells, the loss of E4orf3 function does not appear to exacerbate the defect in viral mRNA transport associated with the loss of E1B-55K.

    Techniques: Infection, Isolation, Mutagenesis

    E1B-55K/E4orf3 double mutant virus directs early protein synthesis to nearly wild-type levels. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 20 PFU per cell. The status of the relevant viral genes (E1B-55K or E4orf3) is indicated below the blots. The levels of E1A, E1B-55K, E2A-72K, E4orf6, and E4orf6/7 proteins from equivalent numbers of cells at 9 h postinfection were determined by Western blotting with primary mouse monoclonal antibodies M73, 2A6, B6-8, and MAb#3, respectively. As controls, the levels of the cellular proteins β-actin and β-tubulin were determined with primary mouse monoclonal antibody clones AC-15 and Tub 2.1, respectively. Sizes are shown to the right (in kilodaltons).

    Journal: Journal of Virology

    Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background

    doi: 10.1128/JVI.78.18.9924-9935.2004

    Figure Lengend Snippet: E1B-55K/E4orf3 double mutant virus directs early protein synthesis to nearly wild-type levels. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 20 PFU per cell. The status of the relevant viral genes (E1B-55K or E4orf3) is indicated below the blots. The levels of E1A, E1B-55K, E2A-72K, E4orf6, and E4orf6/7 proteins from equivalent numbers of cells at 9 h postinfection were determined by Western blotting with primary mouse monoclonal antibodies M73, 2A6, B6-8, and MAb#3, respectively. As controls, the levels of the cellular proteins β-actin and β-tubulin were determined with primary mouse monoclonal antibody clones AC-15 and Tub 2.1, respectively. Sizes are shown to the right (in kilodaltons).

    Article Snippet: Because the cytoplasmic-to-nuclear late mRNA ratio in the double mutant virus-infected cells was the same as in the E1B-55K single mutant virus-infected cells, the loss of E4orf3 function does not appear to exacerbate the defect in viral mRNA transport associated with the loss of E1B-55K.

    Techniques: Mutagenesis, Infection, Western Blot, Clone Assay

    E1B-55K/E4orf3 double mutant virus kills cells more rapidly than the wild-type or parental mutant virus. HeLa cells were infected with the indicated viruses at a multiplicity of 10 PFU per cell. The status of the relevant viral genes is indicated below the histogram. At 72 h postinfection, the fraction of nonviable cells was measured by trypan blue dye uptake. The results shown are the averages of three to five independent infections performed in three independent experiments. Fewer than 1% of the mock-infected cells were trypan blue dye positive at 72 h postinfection. Error bars indicate the standard error of the mean.

    Journal: Journal of Virology

    Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background

    doi: 10.1128/JVI.78.18.9924-9935.2004

    Figure Lengend Snippet: E1B-55K/E4orf3 double mutant virus kills cells more rapidly than the wild-type or parental mutant virus. HeLa cells were infected with the indicated viruses at a multiplicity of 10 PFU per cell. The status of the relevant viral genes is indicated below the histogram. At 72 h postinfection, the fraction of nonviable cells was measured by trypan blue dye uptake. The results shown are the averages of three to five independent infections performed in three independent experiments. Fewer than 1% of the mock-infected cells were trypan blue dye positive at 72 h postinfection. Error bars indicate the standard error of the mean.

    Article Snippet: Because the cytoplasmic-to-nuclear late mRNA ratio in the double mutant virus-infected cells was the same as in the E1B-55K single mutant virus-infected cells, the loss of E4orf3 function does not appear to exacerbate the defect in viral mRNA transport associated with the loss of E1B-55K.

    Techniques: Mutagenesis, Infection

    (A) E1B-55K/E4orf3 double mutant virus-infected cells synthesize viral DNA to the same level as in a wild-type virus infection. HeLa cells were infected with the indicated viruses at a multiplicity of 25 PFU per cell. The status of the E1B-55K and E4orf3 genes in each of the viruses is indicated below the histogram. Total cellular DNA was isolated from equal numbers of adenovirus-infected HeLa cells at 24, 48, and 72 h postinfection, and the amount of viral DNA was measured by hybridization with radioactively labeled adenovirus DNA. The results shown are representative of two to three independent experiments. (B) Viral genomes of E1B-55K/E4orf3 double mutant virus-infected cells form concatemers. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 25 PFU per cell. The status of the E1B-55K and E4 genes in each of the viruses is indicated below the panel. The total DNA present in the cells at 24 h postinfection was visualized by ethidium bromide staining after pulsed-field gel electrophoresis. The sizes of concatemers of 40-kb bacteriophage lambda DNA provided size standards.

    Journal: Journal of Virology

    Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background

    doi: 10.1128/JVI.78.18.9924-9935.2004

    Figure Lengend Snippet: (A) E1B-55K/E4orf3 double mutant virus-infected cells synthesize viral DNA to the same level as in a wild-type virus infection. HeLa cells were infected with the indicated viruses at a multiplicity of 25 PFU per cell. The status of the E1B-55K and E4orf3 genes in each of the viruses is indicated below the histogram. Total cellular DNA was isolated from equal numbers of adenovirus-infected HeLa cells at 24, 48, and 72 h postinfection, and the amount of viral DNA was measured by hybridization with radioactively labeled adenovirus DNA. The results shown are representative of two to three independent experiments. (B) Viral genomes of E1B-55K/E4orf3 double mutant virus-infected cells form concatemers. HeLa cells were mock infected or infected with the indicated viruses at a multiplicity of 25 PFU per cell. The status of the E1B-55K and E4 genes in each of the viruses is indicated below the panel. The total DNA present in the cells at 24 h postinfection was visualized by ethidium bromide staining after pulsed-field gel electrophoresis. The sizes of concatemers of 40-kb bacteriophage lambda DNA provided size standards.

    Article Snippet: Because the cytoplasmic-to-nuclear late mRNA ratio in the double mutant virus-infected cells was the same as in the E1B-55K single mutant virus-infected cells, the loss of E4orf3 function does not appear to exacerbate the defect in viral mRNA transport associated with the loss of E1B-55K.

    Techniques: Mutagenesis, Infection, Isolation, Hybridization, Labeling, Staining, Pulsed-Field Gel, Electrophoresis, Lambda DNA Preparation

    (A) E1B-55K/E4orf3 double mutant virus-infected cells synthesize reduced levels of viral DNA in MO59J and MO59K cells. MO59J and MO59K cells were infected with the indicated viruses at an effective multiplicity of 1 infectious unit per cell. The status of the E1B-55K and E4orf3 genes in each of the viruses is indicated below the histogram. The amount of viral DNA present in equal numbers of adenovirus-infected cells at 48 h postinfection was measured by slot blotting. Representative results from two independent experiments are shown. (B) Viral genomes in E1B-55K/E4orf3 double mutant virus-infected MO59J cells fail to undergo concatemer formation. MO59J and MO59K cells were infected with the wild-type virus and the E1B-55K/E4orf3 double mutant virus at an effective multiplicity of 1 infectious unit per cell, and total infected-cell DNA at 48 h postinfection was analyzed by pulsed-field gel electrophoresis and ethidium bromide staining. (C) The E1B-55K/E4orf3 double mutant virus exhibits a decrease in virus yield in both MO59J and MO59K cells. MO59J and MO59K cells were infected with the indicated viruses, and the virus yield at 48 h postinfection was measured by plaque assay with 293 cells. The status of the relevant viral genes is indicated below the histogram. The results shown are the averages of three independent infections performed in three independent experiments. Error bars indicate the standard error of the mean.

    Journal: Journal of Virology

    Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background

    doi: 10.1128/JVI.78.18.9924-9935.2004

    Figure Lengend Snippet: (A) E1B-55K/E4orf3 double mutant virus-infected cells synthesize reduced levels of viral DNA in MO59J and MO59K cells. MO59J and MO59K cells were infected with the indicated viruses at an effective multiplicity of 1 infectious unit per cell. The status of the E1B-55K and E4orf3 genes in each of the viruses is indicated below the histogram. The amount of viral DNA present in equal numbers of adenovirus-infected cells at 48 h postinfection was measured by slot blotting. Representative results from two independent experiments are shown. (B) Viral genomes in E1B-55K/E4orf3 double mutant virus-infected MO59J cells fail to undergo concatemer formation. MO59J and MO59K cells were infected with the wild-type virus and the E1B-55K/E4orf3 double mutant virus at an effective multiplicity of 1 infectious unit per cell, and total infected-cell DNA at 48 h postinfection was analyzed by pulsed-field gel electrophoresis and ethidium bromide staining. (C) The E1B-55K/E4orf3 double mutant virus exhibits a decrease in virus yield in both MO59J and MO59K cells. MO59J and MO59K cells were infected with the indicated viruses, and the virus yield at 48 h postinfection was measured by plaque assay with 293 cells. The status of the relevant viral genes is indicated below the histogram. The results shown are the averages of three independent infections performed in three independent experiments. Error bars indicate the standard error of the mean.

    Article Snippet: Because the cytoplasmic-to-nuclear late mRNA ratio in the double mutant virus-infected cells was the same as in the E1B-55K single mutant virus-infected cells, the loss of E4orf3 function does not appear to exacerbate the defect in viral mRNA transport associated with the loss of E1B-55K.

    Techniques: Mutagenesis, Infection, Pulsed-Field Gel, Electrophoresis, Staining, Plaque Assay

    E1B-55K/E4orf3 double mutant virus fails to reorganize PML protein. A549 cells were mock infected, infected with the wild-type virus dl 309, the E4orf3 mutant virus dl 341, the E1B-55K mutant virus dl 1520, or the E1B-55K/E4orf3 double mutant virus 55K − /orf3 − at a multiplicity of 10 PFU per cell. At 48 h postinfection, double-label immunofluorescence was used to visualize PML protein (green) with the mouse monoclonal antibody 5E10 and the E4orf3 protein (red) with rat monoclonal antibody 6A11. The merged red and green images are overlaid on a differential interference contrast image of the cell. The micrographs were acquired as a single optical slice with confocal laser scanning microscopy. The inset in each PML image is a twofold enlargement illustrating the characteristic morphology of the indicated PML-containing nuclear bodies.

    Journal: Journal of Virology

    Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background

    doi: 10.1128/JVI.78.18.9924-9935.2004

    Figure Lengend Snippet: E1B-55K/E4orf3 double mutant virus fails to reorganize PML protein. A549 cells were mock infected, infected with the wild-type virus dl 309, the E4orf3 mutant virus dl 341, the E1B-55K mutant virus dl 1520, or the E1B-55K/E4orf3 double mutant virus 55K − /orf3 − at a multiplicity of 10 PFU per cell. At 48 h postinfection, double-label immunofluorescence was used to visualize PML protein (green) with the mouse monoclonal antibody 5E10 and the E4orf3 protein (red) with rat monoclonal antibody 6A11. The merged red and green images are overlaid on a differential interference contrast image of the cell. The micrographs were acquired as a single optical slice with confocal laser scanning microscopy. The inset in each PML image is a twofold enlargement illustrating the characteristic morphology of the indicated PML-containing nuclear bodies.

    Article Snippet: Because the cytoplasmic-to-nuclear late mRNA ratio in the double mutant virus-infected cells was the same as in the E1B-55K single mutant virus-infected cells, the loss of E4orf3 function does not appear to exacerbate the defect in viral mRNA transport associated with the loss of E1B-55K.

    Techniques: Mutagenesis, Infection, Immunofluorescence, Confocal Laser Scanning Microscopy

    Immunoprecipitation of HAV5 E1 proteins from extracts of BHH cell lines with HAV5 E1-specific antibodies. A549, A549-infected with HAV5, 293-Puro, BHH2C, BHH3, and BHH8 cells were labeled with Trans[ 35 S]-label, and cell extracts were immunoprecipitated with anti-HAV2 E1A (13S-5; Santa Cruz) (A), anti-HAV2 E1B-21kDa (3D11; Calbiochem) (B), and anti-HAV5 E1B-55kDa (9C10; Calbiochem) (C) antibodies. Each HAV5 E1 protein is indicated by an arrow. The position of the molecular weight marker is shown on the right side of the panel.

    Journal: Journal of Virology

    Article Title: Development and Characterization of Bovine x Human Hybrid Cell Lines That Efficiently Support the Replication of both Wild-Type Bovine and Human Adenoviruses and Those with E1 Deleted

    doi: 10.1128/JVI.76.12.5882-5892.2002

    Figure Lengend Snippet: Immunoprecipitation of HAV5 E1 proteins from extracts of BHH cell lines with HAV5 E1-specific antibodies. A549, A549-infected with HAV5, 293-Puro, BHH2C, BHH3, and BHH8 cells were labeled with Trans[ 35 S]-label, and cell extracts were immunoprecipitated with anti-HAV2 E1A (13S-5; Santa Cruz) (A), anti-HAV2 E1B-21kDa (3D11; Calbiochem) (B), and anti-HAV5 E1B-55kDa (9C10; Calbiochem) (C) antibodies. Each HAV5 E1 protein is indicated by an arrow. The position of the molecular weight marker is shown on the right side of the panel.

    Article Snippet: HAV5 E1-specific antibodies were purchased as follows: HAV2 E1A (13S-5; Santa Cruz), HAV5 E1B-21kDa (1G11; Calbiochem), and HAV5 E1B-55kDa (9C10; Calbiochem).

    Techniques: Immunoprecipitation, Infection, Labeling, Molecular Weight, Marker

    In vivo regulatory screen of all 6-mers . (a) Left top: naively concatenating three 6-mers creates an oligomer with multiple representatives of ATTGCG (red bars). Center and left: cartoon of a de Bruijn graph. Nodes (colored boxes) represent 6-mers; edges (arrows) represent overlap. A standard de-Bruijn-sequence library is built from one path that traverses each of 4,096 nodes once. Constructed from multiple paths, our MRCC library instead uses one representative for each pair of reverse-complementary 6-mers (green and yellow; self-reverse-complementary palindromes in blue), making it nearly 50% smaller (Additional file 1 ). Right: 16 of 16,384 edges shown. Our algorithm removes reverse-complementary paths (black, red) between palindrome pairs and then decomposes the remaining graph into reverse-complementary cycles. It allowed us to design an ultra-compact library of DNA sequences with uniform 6-mer coverage. (b) Schematic depicting the sub-cloning of each 15-bp multiplexed oligomer into the E1b-Tol2 vector and subsequent injection into single-cell zebrafish embryos. (c) Violin plots depicting the distribution of the expression patterns of each tissue at 24 hpf. White lines indicate the fractional expression values for the empty vector construct. (d) Scatter plot depicting the method by which we selected consistently expressed multiplexed oligomers whose expression was not significantly correlated to minimal-promoter bias. The vertical dotted line denotes the 40% fractional expression threshold that was used, whereas the horizontal dotted line corresponds to a false discovery rate-adjusted P -value of 0.05. (e) Histogram depicting the tissue specificity of the 22 uncorrelated, consistently expressed constructs at 24 hpf. (f) Representative images at 24 hpf for embryos injected with 1EF03 (top) and 3EF09 (bottom), exhibiting broad expression that was correlated with that of the empty vector expression. The full sequence of each construct (with XhoI and BglII flanking sites) is listed below each figure. Both constructs have a 5' GC-rich region. GFP, green fluorescent protein.

    Journal: Genome Biology

    Article Title: A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design

    doi: 10.1186/gb-2013-14-7-r72

    Figure Lengend Snippet: In vivo regulatory screen of all 6-mers . (a) Left top: naively concatenating three 6-mers creates an oligomer with multiple representatives of ATTGCG (red bars). Center and left: cartoon of a de Bruijn graph. Nodes (colored boxes) represent 6-mers; edges (arrows) represent overlap. A standard de-Bruijn-sequence library is built from one path that traverses each of 4,096 nodes once. Constructed from multiple paths, our MRCC library instead uses one representative for each pair of reverse-complementary 6-mers (green and yellow; self-reverse-complementary palindromes in blue), making it nearly 50% smaller (Additional file 1 ). Right: 16 of 16,384 edges shown. Our algorithm removes reverse-complementary paths (black, red) between palindrome pairs and then decomposes the remaining graph into reverse-complementary cycles. It allowed us to design an ultra-compact library of DNA sequences with uniform 6-mer coverage. (b) Schematic depicting the sub-cloning of each 15-bp multiplexed oligomer into the E1b-Tol2 vector and subsequent injection into single-cell zebrafish embryos. (c) Violin plots depicting the distribution of the expression patterns of each tissue at 24 hpf. White lines indicate the fractional expression values for the empty vector construct. (d) Scatter plot depicting the method by which we selected consistently expressed multiplexed oligomers whose expression was not significantly correlated to minimal-promoter bias. The vertical dotted line denotes the 40% fractional expression threshold that was used, whereas the horizontal dotted line corresponds to a false discovery rate-adjusted P -value of 0.05. (e) Histogram depicting the tissue specificity of the 22 uncorrelated, consistently expressed constructs at 24 hpf. (f) Representative images at 24 hpf for embryos injected with 1EF03 (top) and 3EF09 (bottom), exhibiting broad expression that was correlated with that of the empty vector expression. The full sequence of each construct (with XhoI and BglII flanking sites) is listed below each figure. Both constructs have a 5' GC-rich region. GFP, green fluorescent protein.

    Article Snippet: For the concatemeric constructs, 150- to 225-bp regions were synthesized and cloned into E1b-Tol2 (Biomatik USA, LLC, Wilmington, DE, USA).

    Techniques: In Vivo, Sequencing, Construct, Subcloning, Plasmid Preparation, Injection, Expressing

    The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .

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

    Article Title: Influenza virus targets the mRNA export machinery and the nuclear pore complex

    doi: 10.1073/pnas.0610977104

    Figure Lengend Snippet: The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .

    Article Snippet: Immunoblot analysis was performed with anti-Nup98 , anti-E1B-AP5 (Proteintech Group, Chicago, IL), anti-Rae1 , and anti-NXF1/TAP antibodies (BD Transduction Laboratories, San Jose, CA), anti-p15/NXT antibodies (Abnova, Taipei City, Taiwan), mAb414 , and anti-Influenza A antibodies (virions; Biodesign International, Saco, ME).

    Techniques: Incubation, Recombinant, SDS Page, Expressing, Infection, Labeling

    Phenotypic characterization of a stable transgenic line. ( a ) Graphic representation of the DNA constructs used for transgenesis and breeding. ( b ) A 24 h.p.f. embryo expresses Smoa1-EGFP in the forebrain and retinal neuroblasts. A 96 h.p.f. double transgenic embryo derived from crossing of the Tg(krt5:Gal4VP16;14 × UAS:smoa1-EGFP) and the Tg(UAS-E1b:nfsB-mCherry) c264 stable lines expressed GFP ( c ) and mCherry ( d ) in the ONs (arrows). ( e ) A 6-month-old wild-type and ( f ) a transgenic fish with a gross tumor in its left eye (arrow). ( g ) Wild-type fish formed a normal optic chiasm, whereas ( h ) transgenic fish failed to form optic chiasm and showed dramatic expansion of the ONH region (arrow). ( i ) Dissected brain and eyes showing an enlarged ON associating with a gross eye tumor (arrow).

    Journal: Oncogenesis

    Article Title: Activation of Sonic hedgehog signaling in neural progenitor cells promotes glioma development in the zebrafish optic pathway

    doi: 10.1038/oncsis.2014.10

    Figure Lengend Snippet: Phenotypic characterization of a stable transgenic line. ( a ) Graphic representation of the DNA constructs used for transgenesis and breeding. ( b ) A 24 h.p.f. embryo expresses Smoa1-EGFP in the forebrain and retinal neuroblasts. A 96 h.p.f. double transgenic embryo derived from crossing of the Tg(krt5:Gal4VP16;14 × UAS:smoa1-EGFP) and the Tg(UAS-E1b:nfsB-mCherry) c264 stable lines expressed GFP ( c ) and mCherry ( d ) in the ONs (arrows). ( e ) A 6-month-old wild-type and ( f ) a transgenic fish with a gross tumor in its left eye (arrow). ( g ) Wild-type fish formed a normal optic chiasm, whereas ( h ) transgenic fish failed to form optic chiasm and showed dramatic expansion of the ONH region (arrow). ( i ) Dissected brain and eyes showing an enlarged ON associating with a gross eye tumor (arrow).

    Article Snippet: Zebrafish husbandry AB and Tg(UAS-E1b:nfsB-mCherry) c264 strains were acquired from the Zebrafish International Resource Center (ZIRC, Eugene, OR, USA).

    Techniques: Transgenic Assay, Construct, Derivative Assay, Fluorescence In Situ Hybridization

    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

    Journal: EMBO Reports

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

    doi: 10.1038/embor.2009.179

    Figure Lengend Snippet: VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

    Article Snippet: Blower; ); Rae1 and Nup98 for immunoblot ( ); phospho-histone H3 and Ser 10 (Upstate, USA); β-tubulin–fluorescein isothiocyanate (Sigma, USA); hnRNPA1 (M. Matunis); E1B-AP5 (Proteintech Group, USA); pericentrin (Abcam, USA); monoclonal GST (Santa Cruz Biotechnology, USA); polyclonal γ-tubulin (Sigma); and polyclonal VSV ( ).

    Techniques: Incubation, Recombinant, SDS Page, Immunoprecipitation, Immunofluorescence, Microscopy, Polyacrylamide Gel Electrophoresis

    Construction and characterization of viruses . (A) Transcriptional activity of the AFP promoter in different cell lines. The AFP promoter with an SV40 enhancer was incorporated into pGL3-basic. The relative activity of pGL3-SV40EAFP to pGL3-Control in various cell lines are presented as the mean+SD (n = 3). (B) Schematic structure of the viruses Ad.SPDD, Ad.SPDD-HCCS1, and Ad.SPDD-HCCS1. ITR, inverted terminal repeat; ψ, viral packing signal. Ad.SPDD was quadruply modified compared to the wild-type adenovirus; the modifications were the deletion of E1B19K, the deletion of E1B55K, and the deletion of 24 bp of E1A, as well as deletion of the survivin promoter controlling E1A expression. HCCS1 or HA-tagged HCCS1 in a cassette was carried by Ad.SPDD. (C) Characterization of viruses by western blot analysis. Huh-7 cells were infected with the indicated viruses, and the levels of the expressed E1A, E1B 19KDa, E1B 55KDa and HA-Tag were analyzed 48 hours post infection (h.p.i), with actin as loading control. (***: P

    Journal: Molecular Cancer

    Article Title: HCCS1-armed, quadruple-regulated oncolytic adenovirus specific for liver cancer as a cancer targeting gene-viro-therapy strategy

    doi: 10.1186/1476-4598-10-133

    Figure Lengend Snippet: Construction and characterization of viruses . (A) Transcriptional activity of the AFP promoter in different cell lines. The AFP promoter with an SV40 enhancer was incorporated into pGL3-basic. The relative activity of pGL3-SV40EAFP to pGL3-Control in various cell lines are presented as the mean+SD (n = 3). (B) Schematic structure of the viruses Ad.SPDD, Ad.SPDD-HCCS1, and Ad.SPDD-HCCS1. ITR, inverted terminal repeat; ψ, viral packing signal. Ad.SPDD was quadruply modified compared to the wild-type adenovirus; the modifications were the deletion of E1B19K, the deletion of E1B55K, and the deletion of 24 bp of E1A, as well as deletion of the survivin promoter controlling E1A expression. HCCS1 or HA-tagged HCCS1 in a cassette was carried by Ad.SPDD. (C) Characterization of viruses by western blot analysis. Huh-7 cells were infected with the indicated viruses, and the levels of the expressed E1A, E1B 19KDa, E1B 55KDa and HA-Tag were analyzed 48 hours post infection (h.p.i), with actin as loading control. (***: P

    Article Snippet: The E1B 55kDa-specific antibody was purchased from Oncogene (Cambridge, MA), the E1B 19kDa-specific antibody was purchased from Calbiochem (San Diego, CA), and the Hexona-specific antibody was obtained from Millipore (Billerica, MA).

    Techniques: Activity Assay, Modification, Expressing, Western Blot, Infection

    (A) Localization of E1B-AP5, ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.

    Journal: Journal of Virology

    Article Title: A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

    doi: 10.1128/JVI.00170-08

    Figure Lengend Snippet: (A) Localization of E1B-AP5, ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.

    Article Snippet: Small interfering RNA (siRNA) oligonucleotides targeting ATR and E1B-AP5 were purchased from Ambion, and their sequences were as follows: 5′-GGAAUAUAAUACAGUUGUATT-3′ (ATR) and 5′-GCAGUGGAACCAGUACUAUTT-3′ (E1B-AP5).

    Techniques: Infection, Microscopy, Staining

    Effects of Ad infection on protein levels of E1B-55K binding proteins. A549 cells were infected with 10 PFU per cell of either wt Ad5 (A) or wt Ad12 (B). Cells were harvested at the appropriate times postinfection, and 50 μg protein samples was separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were then Western blotted for E1B-AP5 (i), MRE11 (ii), p53 (iii), E1B-55K (iv), and β-actin (v) using the appropriate antibodies and visualized by enhanced chemiluminescence. (C) Immunofluorescent Zeiss LSM510-Meta confocal microscopic detection of E1B-AP5 protein expression in wt Ad5- and wt Ad12-infected A549 cells. Cells were infected with either 10 PFU/cell wt Ad5 or wt Ad12, subsequently fixed with 4% (wt/vol) paraformaldehyde in PBS, and permeabilized with acetone 24 h postinfection. E1B-AP5 (i and iv), Ad5 DBP (ii), and Ad12 E1B-55K (v) were then visualized using the appropriate antibodies. Nuclei are stained with DAPI and are shown in blue.

    Journal: Journal of Virology

    Article Title: A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

    doi: 10.1128/JVI.00170-08

    Figure Lengend Snippet: Effects of Ad infection on protein levels of E1B-55K binding proteins. A549 cells were infected with 10 PFU per cell of either wt Ad5 (A) or wt Ad12 (B). Cells were harvested at the appropriate times postinfection, and 50 μg protein samples was separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were then Western blotted for E1B-AP5 (i), MRE11 (ii), p53 (iii), E1B-55K (iv), and β-actin (v) using the appropriate antibodies and visualized by enhanced chemiluminescence. (C) Immunofluorescent Zeiss LSM510-Meta confocal microscopic detection of E1B-AP5 protein expression in wt Ad5- and wt Ad12-infected A549 cells. Cells were infected with either 10 PFU/cell wt Ad5 or wt Ad12, subsequently fixed with 4% (wt/vol) paraformaldehyde in PBS, and permeabilized with acetone 24 h postinfection. E1B-AP5 (i and iv), Ad5 DBP (ii), and Ad12 E1B-55K (v) were then visualized using the appropriate antibodies. Nuclei are stained with DAPI and are shown in blue.

    Article Snippet: Small interfering RNA (siRNA) oligonucleotides targeting ATR and E1B-AP5 were purchased from Ambion, and their sequences were as follows: 5′-GGAAUAUAAUACAGUUGUATT-3′ (ATR) and 5′-GCAGUGGAACCAGUACUAUTT-3′ (E1B-AP5).

    Techniques: Infection, Binding Assay, SDS Page, Western Blot, Expressing, Staining

    Colocalization of E1B-AP5 and RPA32, and ATRIP and RPA32, at Ad5 (A) and Ad12 (B) replication centers. Cells were infected with 10 PFU/cell of the appropriate virus. Ad5-infected and Ad12-infected cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h and 48 h postinfection, respectively. E1B-AP5, RPA32, and ATRIP localizations were visualized using the appropriate antibodies. Colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Regions of substantial colocalization are shown as yellow in the merged image. Nuclei are stained with DAPI and are shown in blue.

    Journal: Journal of Virology

    Article Title: A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

    doi: 10.1128/JVI.00170-08

    Figure Lengend Snippet: Colocalization of E1B-AP5 and RPA32, and ATRIP and RPA32, at Ad5 (A) and Ad12 (B) replication centers. Cells were infected with 10 PFU/cell of the appropriate virus. Ad5-infected and Ad12-infected cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h and 48 h postinfection, respectively. E1B-AP5, RPA32, and ATRIP localizations were visualized using the appropriate antibodies. Colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Regions of substantial colocalization are shown as yellow in the merged image. Nuclei are stained with DAPI and are shown in blue.

    Article Snippet: Small interfering RNA (siRNA) oligonucleotides targeting ATR and E1B-AP5 were purchased from Ambion, and their sequences were as follows: 5′-GGAAUAUAAUACAGUUGUATT-3′ (ATR) and 5′-GCAGUGGAACCAGUACUAUTT-3′ (E1B-AP5).

    Techniques: Infection, Microscopy, Staining

    (A) Colocalization of E1B-AP5 with E1B-55K at Ad5 and Ad12 replication centers. Cells were infected with 10 PFU/cell of the appropriate virus. Infected cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h postinfection. E1B-AP5 and Ad E1B-55K were visualized using the appropriate antibodies. (B) Recruitment of E1B-AP5, RPA32, and ATRIP to Ad5 replication centers is independent of E1B-55K. Cells were infected with 10 PFU/cell of Ad5 dl 1520 and treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h postinfection. E1B-AP5, Ad5 DBP, RPA32, and ATRIP were visualized using the appropriate antibodies. Colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Regions of substantial colocalization are shown as yellow in the merged image. Nuclei are stained with DAPI and are shown in blue.

    Journal: Journal of Virology

    Article Title: A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

    doi: 10.1128/JVI.00170-08

    Figure Lengend Snippet: (A) Colocalization of E1B-AP5 with E1B-55K at Ad5 and Ad12 replication centers. Cells were infected with 10 PFU/cell of the appropriate virus. Infected cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h postinfection. E1B-AP5 and Ad E1B-55K were visualized using the appropriate antibodies. (B) Recruitment of E1B-AP5, RPA32, and ATRIP to Ad5 replication centers is independent of E1B-55K. Cells were infected with 10 PFU/cell of Ad5 dl 1520 and treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h postinfection. E1B-AP5, Ad5 DBP, RPA32, and ATRIP were visualized using the appropriate antibodies. Colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Regions of substantial colocalization are shown as yellow in the merged image. Nuclei are stained with DAPI and are shown in blue.

    Article Snippet: Small interfering RNA (siRNA) oligonucleotides targeting ATR and E1B-AP5 were purchased from Ambion, and their sequences were as follows: 5′-GGAAUAUAAUACAGUUGUATT-3′ (ATR) and 5′-GCAGUGGAACCAGUACUAUTT-3′ (E1B-AP5).

    Techniques: Infection, Microscopy, Staining

    (A) Ad5 and Ad12 differentially regulate the phosphorylation of RPA32, Rad9, and Smc1 during infection. A549 cells were infected with 10 PFU/cell of either wt Ad5 or wt Ad12. Cells were harvested at the appropriate times postinfection, and 50 μg protein samples was separated by SDS-PAGE. After electrophoretic transfer onto nitrocellulose, membranes were Western blotted for RPA32 (i), RPA32 S4/8 (ii), Rad9 (iii), Smc1-S966 (iv), Smc1 (v), Chk1-S345 (vi), Chk1 (vii), γ-H2AX (viii), and H2AX (ix) using the appropriate antibodies. (B) E1B-AP5 is required for Ad12-induced phosphorylation of RPA32. A549 cells were initially treated with either nonsilencing (non-sil.) siRNA or siRNA oligonucleotides specific for the E1B-AP5 gene. Cells were subsequently infected with either Ad5 or Ad12 (at 10 PFU/cell), and whole-cell lysates were prepared at the appropriate times postinfection. After SDS-PAGE and transfer onto nitrocellulose, membranes were probed for E1B-AP5 (i), RPA32 (ii), RPA32 S4/8 (iii), Smc1-S966 (iv), Smc1 (v), γ-H2AX (vi), and H2AX (vii) with the appropriate antibodies. Antigens were visualized by ECL.

    Journal: Journal of Virology

    Article Title: A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

    doi: 10.1128/JVI.00170-08

    Figure Lengend Snippet: (A) Ad5 and Ad12 differentially regulate the phosphorylation of RPA32, Rad9, and Smc1 during infection. A549 cells were infected with 10 PFU/cell of either wt Ad5 or wt Ad12. Cells were harvested at the appropriate times postinfection, and 50 μg protein samples was separated by SDS-PAGE. After electrophoretic transfer onto nitrocellulose, membranes were Western blotted for RPA32 (i), RPA32 S4/8 (ii), Rad9 (iii), Smc1-S966 (iv), Smc1 (v), Chk1-S345 (vi), Chk1 (vii), γ-H2AX (viii), and H2AX (ix) using the appropriate antibodies. (B) E1B-AP5 is required for Ad12-induced phosphorylation of RPA32. A549 cells were initially treated with either nonsilencing (non-sil.) siRNA or siRNA oligonucleotides specific for the E1B-AP5 gene. Cells were subsequently infected with either Ad5 or Ad12 (at 10 PFU/cell), and whole-cell lysates were prepared at the appropriate times postinfection. After SDS-PAGE and transfer onto nitrocellulose, membranes were probed for E1B-AP5 (i), RPA32 (ii), RPA32 S4/8 (iii), Smc1-S966 (iv), Smc1 (v), γ-H2AX (vi), and H2AX (vii) with the appropriate antibodies. Antigens were visualized by ECL.

    Article Snippet: Small interfering RNA (siRNA) oligonucleotides targeting ATR and E1B-AP5 were purchased from Ambion, and their sequences were as follows: 5′-GGAAUAUAAUACAGUUGUATT-3′ (ATR) and 5′-GCAGUGGAACCAGUACUAUTT-3′ (E1B-AP5).

    Techniques: Infection, SDS Page, Western Blot

    Clusters of coregulated cellular RNAs 30 h after infection with Ad5 or E1B mutants. The expression profiles of significantly regulated genes were clustered into eight distinct groups with similar expression response profiles across the different Ad5 genotypes as described in Materials and Methods. Each cluster was searched for statistically significant enrichment of genes that code for proteins with common functions ( P values are Bonferroni corrected for multiple hypothesis testing). GO terms enriched in clusters 2, 3, and 4 are shown at right, with the corresponding P values. Genes that increased and decreased in expression are shown in yellow and blue, respectively, as indicated on the scale bar. hpi, hours postinfection.

    Journal: Journal of Virology

    Article Title: The Adenoviral E1B 55-Kilodalton Protein Controls Expression of Immune Response Genes but Not p53-Dependent Transcription ▿

    doi: 10.1128/JVI.02269-08

    Figure Lengend Snippet: Clusters of coregulated cellular RNAs 30 h after infection with Ad5 or E1B mutants. The expression profiles of significantly regulated genes were clustered into eight distinct groups with similar expression response profiles across the different Ad5 genotypes as described in Materials and Methods. Each cluster was searched for statistically significant enrichment of genes that code for proteins with common functions ( P values are Bonferroni corrected for multiple hypothesis testing). GO terms enriched in clusters 2, 3, and 4 are shown at right, with the corresponding P values. Genes that increased and decreased in expression are shown in yellow and blue, respectively, as indicated on the scale bar. hpi, hours postinfection.

    Article Snippet: Plasmids carrying the desired mutations were identified by the presence of a new restriction endonuclease site, and the presence of the mutations was confirmed by sequencing the E1B gene (Genewiz Inc.).

    Techniques: Infection, Expressing

    Effects of the sub17 mutations on accumulation of the E1B 55-kDa protein and protein V. (A) The coding sequences of the E1B proteins are shown to scale below the arrow representing the major E1B mRNA species, with different reading frames in white, gray, and black. As summarized above the mRNA, deletion of bp 2437 in the Hr6 genomes results in a shift of reading frame and termination of translation a short distance downstream. The C-terminal sequences of the wild-type and sub17 E1B 55-kDa proteins are shown between the expansion lines below, with the substitutions indicated in bold. (B) The E1B 55-kDa protein was examined 36 h after infection with Ad5 or Ad5E1B-sub17 (S17) or in mock-infected cells (M) by immunoblotting as described in Materials and Methods. Numbers at left are molecular masses in kilodaltons.

    Journal: Journal of Virology

    Article Title: The Adenoviral E1B 55-Kilodalton Protein Controls Expression of Immune Response Genes but Not p53-Dependent Transcription ▿

    doi: 10.1128/JVI.02269-08

    Figure Lengend Snippet: Effects of the sub17 mutations on accumulation of the E1B 55-kDa protein and protein V. (A) The coding sequences of the E1B proteins are shown to scale below the arrow representing the major E1B mRNA species, with different reading frames in white, gray, and black. As summarized above the mRNA, deletion of bp 2437 in the Hr6 genomes results in a shift of reading frame and termination of translation a short distance downstream. The C-terminal sequences of the wild-type and sub17 E1B 55-kDa proteins are shown between the expansion lines below, with the substitutions indicated in bold. (B) The E1B 55-kDa protein was examined 36 h after infection with Ad5 or Ad5E1B-sub17 (S17) or in mock-infected cells (M) by immunoblotting as described in Materials and Methods. Numbers at left are molecular masses in kilodaltons.

    Article Snippet: Plasmids carrying the desired mutations were identified by the presence of a new restriction endonuclease site, and the presence of the mutations was confirmed by sequencing the E1B gene (Genewiz Inc.).

    Techniques: Infection

    Immunoprecipitation of HAV5 E1 proteins from extracts of BHH cell lines with HAV5 E1-specific antibodies. A549, A549-infected with HAV5, 293-Puro, BHH2C, BHH3, and BHH8 cells were labeled with Trans[ 35 S]-label, and cell extracts were immunoprecipitated with anti-HAV2 E1A (13S-5; Santa Cruz) (A), anti-HAV2 E1B-21kDa (3D11; Calbiochem) (B), and anti-HAV5 E1B-55kDa (9C10; Calbiochem) (C) antibodies. Each HAV5 E1 protein is indicated by an arrow. The position of the molecular weight marker is shown on the right side of the panel.

    Journal: Journal of Virology

    Article Title: Development and Characterization of Bovine x Human Hybrid Cell Lines That Efficiently Support the Replication of both Wild-Type Bovine and Human Adenoviruses and Those with E1 Deleted

    doi: 10.1128/JVI.76.12.5882-5892.2002

    Figure Lengend Snippet: Immunoprecipitation of HAV5 E1 proteins from extracts of BHH cell lines with HAV5 E1-specific antibodies. A549, A549-infected with HAV5, 293-Puro, BHH2C, BHH3, and BHH8 cells were labeled with Trans[ 35 S]-label, and cell extracts were immunoprecipitated with anti-HAV2 E1A (13S-5; Santa Cruz) (A), anti-HAV2 E1B-21kDa (3D11; Calbiochem) (B), and anti-HAV5 E1B-55kDa (9C10; Calbiochem) (C) antibodies. Each HAV5 E1 protein is indicated by an arrow. The position of the molecular weight marker is shown on the right side of the panel.

    Article Snippet: HAV5 E1-specific antibodies were purchased as follows: HAV2 E1A (13S-5; Santa Cruz), HAV5 E1B-21kDa (1G11; Calbiochem), and HAV5 E1B-55kDa (9C10; Calbiochem).

    Techniques: Immunoprecipitation, Infection, Labeling, Molecular Weight, Marker

    Reduced E1B 20K expression correlated with loss of Ad14p1 CPE corpse repression of NF-κB activation. (A) Immunoblot analysis of E1B 19/20K expression in A549 cells infected with wt Ad14, wt Ad5, or Ad14p1 clinical isolate 1986T. (B) RT-qPCR analysis

    Journal: Journal of Virology

    Article Title: Low-Level Expression of the E1B 20-Kilodalton Protein by Adenovirus 14p1 Enhances Viral Immunopathogenesis

    doi: 10.1128/JVI.01790-15

    Figure Lengend Snippet: Reduced E1B 20K expression correlated with loss of Ad14p1 CPE corpse repression of NF-κB activation. (A) Immunoblot analysis of E1B 19/20K expression in A549 cells infected with wt Ad14, wt Ad5, or Ad14p1 clinical isolate 1986T. (B) RT-qPCR analysis

    Article Snippet: Actin (Sigma-Aldrich antibody) and Ad14 E1B 20K (mouse anti-Ad2 E1B 19K monoclonal antibody, 3D11; Calbiochem) were detected by immunoblotting using enhanced chemiluminescence (ECL).

    Techniques: Expressing, Activation Assay, Infection, Quantitative RT-PCR

    Construction and characterization of the E1B 55-kDa- and E4 Orf3-null double-mutant virus AdEasyE1Δ2347-dl341. (A) The origins of the segments of the double mutant virus genome are shown schematically, indicating the positions of the single-pair

    Journal: Journal of Virology

    Article Title: Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

    doi: 10.1128/JVI.03072-14

    Figure Lengend Snippet: Construction and characterization of the E1B 55-kDa- and E4 Orf3-null double-mutant virus AdEasyE1Δ2347-dl341. (A) The origins of the segments of the double mutant virus genome are shown schematically, indicating the positions of the single-pair

    Article Snippet: To assay for the E4 mutation, the region of the genome between bp 34353 and 34382 was amplified by PCR of viral DNAs containing the E1B mutation and sequenced (Genewiz).

    Techniques: Mutagenesis

    HCMV IE1 and IE2 genes and their products are not present in transformed BRK cell lines. ( A ) Lack of expression of the IE1 or IE2 protein in transformed BRK cell lines when assayed by immunoprecipitation of 35 S-labeled cell extracts using monoclonal antibody mAB810. A-1 is a cell line established by transfection with the E1A-expressing plasmid alone. A/1/2–1 to A/1/2–10 are cell lines transformed by E1A plus IE1 and IE2. Lanes designated HCMV INF display immunoprecipitates from HCMV-infected human foreskin fibroblasts; lane 13 is an exposure 1/8 as long as that presented for lanes 1–12. Numbers to the left of the gel mark the positions of marker proteins whose sizes are indicated in kDa. ( B ) Southern blot analysis showing the lack of IE1 - or IE2 -specific DNA in E1A/IE1/IE2-induced transformants (lanes 1–10). A/B-1 and A/B-2 are cell lines transformed with E1A and E1B-19-kDa. HeLa, 293, and HP1.14 (a derivative HeLa cells expressing IE1) are included as controls. pCGN-IE1 and pCGN-IE2 are IE1 and IE2 expression plasmids, respectively, in amounts equivalent to the rest of the samples, assuming they contain one copy of each of the HCMV genes per cell. Numbers to the left of the gel are marker DNA sizes in kb. ( C ) Expression of E1A protein as determined by immunoprecipitation followed by immunoblot analysis, both with monoclonal antibody M73 to E1A. Lanes are labeled as in A and B . Numbers to the left of the gel are marker protein sizes in kDa. Autoradiograms were scanned and cropped using Photoshop and figures were prepared using Freehand software.

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

    Article Title: Human cytomegalovirus IE1 and IE2 proteins are mutagenic and mediate "hit-and-run" oncogenic transformation in cooperation with the adenovirus E1A proteins

    doi:

    Figure Lengend Snippet: HCMV IE1 and IE2 genes and their products are not present in transformed BRK cell lines. ( A ) Lack of expression of the IE1 or IE2 protein in transformed BRK cell lines when assayed by immunoprecipitation of 35 S-labeled cell extracts using monoclonal antibody mAB810. A-1 is a cell line established by transfection with the E1A-expressing plasmid alone. A/1/2–1 to A/1/2–10 are cell lines transformed by E1A plus IE1 and IE2. Lanes designated HCMV INF display immunoprecipitates from HCMV-infected human foreskin fibroblasts; lane 13 is an exposure 1/8 as long as that presented for lanes 1–12. Numbers to the left of the gel mark the positions of marker proteins whose sizes are indicated in kDa. ( B ) Southern blot analysis showing the lack of IE1 - or IE2 -specific DNA in E1A/IE1/IE2-induced transformants (lanes 1–10). A/B-1 and A/B-2 are cell lines transformed with E1A and E1B-19-kDa. HeLa, 293, and HP1.14 (a derivative HeLa cells expressing IE1) are included as controls. pCGN-IE1 and pCGN-IE2 are IE1 and IE2 expression plasmids, respectively, in amounts equivalent to the rest of the samples, assuming they contain one copy of each of the HCMV genes per cell. Numbers to the left of the gel are marker DNA sizes in kb. ( C ) Expression of E1A protein as determined by immunoprecipitation followed by immunoblot analysis, both with monoclonal antibody M73 to E1A. Lanes are labeled as in A and B . Numbers to the left of the gel are marker protein sizes in kDa. Autoradiograms were scanned and cropped using Photoshop and figures were prepared using Freehand software.

    Article Snippet: For the two sets of coverslips collected from cells transfected with vectors expressing the E1A plus E1B-19-kDa proteins, one set was stained with anti-E1A antibody (M73), and the other was stained with monoclonal antibody to E1B-19-kDa (Oncogene Science).

    Techniques: Transformation Assay, Expressing, Immunoprecipitation, Labeling, Transfection, Plasmid Preparation, Infection, Marker, Southern Blot, Software

    The IE1 and IE2 gene products are expressed transiently during the transformation of BRK cells. BRK cells seeded on coverslips were transfected with expression plasmids for E1A and E1B-19-kDa ( Left ) or plasmids for E1A and IE1 and IE2 ( Right ). At 2, 5, 13, and 19 days after transfection, two coverslips from each transfection were fixed and kept in phosphate-buffered saline until coverslips for all time points were collected. Expression of the transfected genes was examined by indirect immunofluorescent staining with appropriate monoclonal antibodies. Anti-E1A, anti-E1B-19-kDa, and anti-IE1/2 identify the antibodies used for the immunofluorescent staining. Images are from independent coverslips. The images from days 13 and 19 display transformed foci whose cells were positive for E1A and E1B-19-kDa proteins, but completely negative for IE1 and IE2 protein expression. (×50).

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

    Article Title: Human cytomegalovirus IE1 and IE2 proteins are mutagenic and mediate "hit-and-run" oncogenic transformation in cooperation with the adenovirus E1A proteins

    doi:

    Figure Lengend Snippet: The IE1 and IE2 gene products are expressed transiently during the transformation of BRK cells. BRK cells seeded on coverslips were transfected with expression plasmids for E1A and E1B-19-kDa ( Left ) or plasmids for E1A and IE1 and IE2 ( Right ). At 2, 5, 13, and 19 days after transfection, two coverslips from each transfection were fixed and kept in phosphate-buffered saline until coverslips for all time points were collected. Expression of the transfected genes was examined by indirect immunofluorescent staining with appropriate monoclonal antibodies. Anti-E1A, anti-E1B-19-kDa, and anti-IE1/2 identify the antibodies used for the immunofluorescent staining. Images are from independent coverslips. The images from days 13 and 19 display transformed foci whose cells were positive for E1A and E1B-19-kDa proteins, but completely negative for IE1 and IE2 protein expression. (×50).

    Article Snippet: For the two sets of coverslips collected from cells transfected with vectors expressing the E1A plus E1B-19-kDa proteins, one set was stained with anti-E1A antibody (M73), and the other was stained with monoclonal antibody to E1B-19-kDa (Oncogene Science).

    Techniques: Transformation Assay, Transfection, Expressing, Staining

    Effect of overexpression and siRNA knockdown of NRF2 on E1b expression

    Journal: Biochimica et biophysica acta

    Article Title: Intronic DNA elements regulate Nrf-2 chemical responsiveness of the human microsomal epoxide hydrolase gene (EPHX1) through a far upstream alternative promoter

    doi: 10.1016/j.bbagrm.2014.03.014

    Figure Lengend Snippet: Effect of overexpression and siRNA knockdown of NRF2 on E1b expression

    Article Snippet: Therefore, although the factors contributing to the E1b chromatin looping remain to be elucidated, they represent unique targets as E1b expression modulators that may in turn be regulated through the action of xenobiotics at this distal enhancer.

    Techniques: Over Expression, Expressing

    Identification of regulatory elements involved in E1b induction

    Journal: Biochimica et biophysica acta

    Article Title: Intronic DNA elements regulate Nrf-2 chemical responsiveness of the human microsomal epoxide hydrolase gene (EPHX1) through a far upstream alternative promoter

    doi: 10.1016/j.bbagrm.2014.03.014

    Figure Lengend Snippet: Identification of regulatory elements involved in E1b induction

    Article Snippet: Therefore, although the factors contributing to the E1b chromatin looping remain to be elucidated, they represent unique targets as E1b expression modulators that may in turn be regulated through the action of xenobiotics at this distal enhancer.

    Techniques:

    Role of DNase HS sites in Nrf2-mediated induction of E1b

    Journal: Biochimica et biophysica acta

    Article Title: Intronic DNA elements regulate Nrf-2 chemical responsiveness of the human microsomal epoxide hydrolase gene (EPHX1) through a far upstream alternative promoter

    doi: 10.1016/j.bbagrm.2014.03.014

    Figure Lengend Snippet: Role of DNase HS sites in Nrf2-mediated induction of E1b

    Article Snippet: Therefore, although the factors contributing to the E1b chromatin looping remain to be elucidated, they represent unique targets as E1b expression modulators that may in turn be regulated through the action of xenobiotics at this distal enhancer.

    Techniques:

    Antioxidants induce E1b expression

    Journal: Biochimica et biophysica acta

    Article Title: Intronic DNA elements regulate Nrf-2 chemical responsiveness of the human microsomal epoxide hydrolase gene (EPHX1) through a far upstream alternative promoter

    doi: 10.1016/j.bbagrm.2014.03.014

    Figure Lengend Snippet: Antioxidants induce E1b expression

    Article Snippet: Therefore, although the factors contributing to the E1b chromatin looping remain to be elucidated, they represent unique targets as E1b expression modulators that may in turn be regulated through the action of xenobiotics at this distal enhancer.

    Techniques: Expressing

    REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals (pG5 [reporter plasmid pG5 E1b Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.

    Journal: Journal of Virology

    Article Title: Expression of Human Herpesvirus 6B rep within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo

    doi:

    Figure Lengend Snippet: REP binds hTBP in vivo as determined by GAL4 two-hybrid system. All of the negative controls resulted in relatively low signals (pG5 [reporter plasmid pG5 E1b Luc] + pM [GAL4 DNA binding domain-based expression plasmid] + pVP16 [HSV VP16 transactivation domain-based expression plasmid], pG5 + pMhTBP + pVP16, pG5 + pM + pVP16rep or pG5). In contrast, both the positive control (pG5 + pM53 [p53]; + pVP16-T [large T]) and pG5 + PMhTBP + pVP16rep gave significantly higher signals. Furthermore, pVP16 RepΔEco+pMhTBP showed higher luciferase activity than pVP16 Rep+pMhTBP. On the other hand, pVP RepΔMlu showed much higher activity than either pVP16 Rep or pVP16 RepΔEco. Thus, these data are consistent with a significant REP-TBP interaction in vivo. Data (means + standard deviations [error bars] of the relative luciferase activity) were calculated from triplicate cultures and are representative of three independent experiments.

    Article Snippet: A reporter plasmid, pG5 E1b Luc, which was generated by replacing the chloramphenicol acetyltransferase gene in pG5 E1b CAT (Clontech) with the firefly luciferase gene was transfected at 30 ng/well with either 0.3 μg of the pM-based or the pVP-based expression vector per well.

    Techniques: In Vivo, Plasmid Preparation, Binding Assay, Expressing, Positive Control, Luciferase, Activity Assay