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Millipore e1b 55k
E1b 55k, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species ▿Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species ▿ †
Article Snippet: .. After being blocked for 1 h at room temperature (RT) with a PBS-milk solution (PBS, 0.05% Tween 20, 5% nonfat dry milk) or Tris-buffered saline (TBS)-casein solution (TBS, 0.1% Tween 20, 2% casein), in the case of phospho-Ser139 H2AX, membranes were incubated overnight at 4°C with primary antibodies specific for the following: NS1 (rabbit antiserum anti-SP8, dilution of 1:5,000) , N-terminal NS1 and NS2 (1:3,000) , NS2 (rabbit antiserum anti-SP6, 1:1,000) , β-tubulin (1:5,000; Sigma), E1A (1:1,000; BD Pharmingen), E1B-55K (1:10; a generous gift from Thomas Dobner, Heinrich-Pette-Institute, Hamburg, Germany), E1B-19K (1:1,000; Calbiochem), α-actin (1:10,000; MP Biomedicals), poly(ADP-ribose) polymerase (PARP) and pRB (1:1,000; BD Pharmingen), caspase 9 (1:1,000; Cell Signaling), caspase 3 (1:1,000; Stressgen), cyclin A, cyclin B1, p107, p130, p21, p27, CDC II (1:1,000; Santa Cruz Biotechnology), p53 (1:1,000; Oncogene), and phospho-Ser139 histone H2AX (γ-H2AX) (1:3,000; Lake Placid Biologicals). .. After the membranes were washed, peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (1:5,000; Santa Cruz Biotechnology) were added for 1 h at RT.

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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 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e1b 55k/product/Millipore
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e1b 55k - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    85
    Millipore anti e1b 55k antibody 9c10
    Cytolytic activity in tumor cells. DU145 and U2OS cells were seeded into 96-well plates at a density of 2.5 × 10 3 cells/well. Twenty-four hours after seeding, cells were infected with serial threefold dilutions of <t>E1B-55K</t> mutant viruses, ranging from an MOI of 30 to an MOI of 1.5 × 10 −3 . dl 309 and ONYX-015 were included as controls. MTT assays were performed 6 days after infection as described in Materials and Methods. The MOIs that resulted in 50% cell killing were defined as IC50 and were plotted for each virus. Results from one of the two independent experiments are shown here. Similar results were obtained in the other experiment.
    Anti E1b 55k Antibody 9c10, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti e1b 55k antibody 9c10/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti e1b 55k antibody 9c10 - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    85
    Millipore anti ad5 e1b 55k rat monoclonal antibody
    Conformational changes in Bak and Bax and Bak-Bax interaction in <t>Ad5</t> dl 337- and Ad5E1B − -infected HeLa cells. (A) Immunoprecipitation (IP) of Bak from mock-, Ad5 dl 309-, Ad5 dl 337-, and Ad5E1B − -infected cells. Immunoprecipitations were carried out with anti-BRCA-2, anti-Bak(23-37), anti-Bak(Ab-1), and anti-Bak(−TM) antibodies (Abs) from the soluble fraction of cells lysed in CHAPS-containing buffer at 24 and 48 h postinfection. Ad5 dl 309-infected HeLa cell lysate (M) was used as a marker for Bak, Bax, and <t>E1B</t> 19K. Western blotting was carried out on precipitated material with a combination of anti-Bak and anti-Bax antibodies (top four panels) or with anti-E1B 19K antibody (bottom four panels). (B) Immunoprecipitation of Bax from mock-, Ad5 dl 309-, Ad5 dl 337-, and Ad5E1B − -infected cells. Experiment was executed as for panel A except that immunoprecipitation was carried out with anti-Myc, anti-Bax(11-30), anti-Bax(150-165), and anti-Bax(43-61) antibodies. Western blotting was carried out as for panel A.
    Anti Ad5 E1b 55k Rat Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ad5 e1b 55k rat monoclonal antibody/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ad5 e1b 55k rat monoclonal antibody - by Bioz Stars, 2020-09
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    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

    Cytolytic activity in tumor cells. DU145 and U2OS cells were seeded into 96-well plates at a density of 2.5 × 10 3 cells/well. Twenty-four hours after seeding, cells were infected with serial threefold dilutions of E1B-55K mutant viruses, ranging from an MOI of 30 to an MOI of 1.5 × 10 −3 . dl 309 and ONYX-015 were included as controls. MTT assays were performed 6 days after infection as described in Materials and Methods. The MOIs that resulted in 50% cell killing were defined as IC50 and were plotted for each virus. Results from one of the two independent experiments are shown here. Similar results were obtained in the other experiment.

    Journal: Journal of Virology

    Article Title: Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein

    doi: 10.1128/JVI.75.9.4297-4307.2001

    Figure Lengend Snippet: Cytolytic activity in tumor cells. DU145 and U2OS cells were seeded into 96-well plates at a density of 2.5 × 10 3 cells/well. Twenty-four hours after seeding, cells were infected with serial threefold dilutions of E1B-55K mutant viruses, ranging from an MOI of 30 to an MOI of 1.5 × 10 −3 . dl 309 and ONYX-015 were included as controls. MTT assays were performed 6 days after infection as described in Materials and Methods. The MOIs that resulted in 50% cell killing were defined as IC50 and were plotted for each virus. Results from one of the two independent experiments are shown here. Similar results were obtained in the other experiment.

    Article Snippet: All antibodies were diluted in the permeabilization and blocking solution. p53 antibody DO-1 (Calbiochem) was diluted 1:100, anti-E1B-55K antibody 9C10 (Calbiochem) was diluted 1:100, and the rabbit anti-E4orf6 antibody 1807-3 was diluted 1:500.

    Techniques: Activity Assay, Infection, Mutagenesis, MTT Assay

    Effects of E1B-55K mutations on p53 accumulation and viral gene expression. A549 cells were either mock infected (mock) or infected with dl 309, WtD, ONYX-015, or viruses harboring various E1B-55K mutants. All infections were performed at an MOI of 10. Cell extracts were prepared at 24 h postinfection and were separated by SDS-PAGE. Steady-state levels of E1B-55K mutants, p53, E2A, and fiber were determined by Western blotting with monoclonal antibodies 2A6, DO-1, B6-6, and a rabbit polyclonal anti-fiber antibody, respectively. Blots were visualized with ECL, as described in Materials and Methods.

    Journal: Journal of Virology

    Article Title: Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein

    doi: 10.1128/JVI.75.9.4297-4307.2001

    Figure Lengend Snippet: Effects of E1B-55K mutations on p53 accumulation and viral gene expression. A549 cells were either mock infected (mock) or infected with dl 309, WtD, ONYX-015, or viruses harboring various E1B-55K mutants. All infections were performed at an MOI of 10. Cell extracts were prepared at 24 h postinfection and were separated by SDS-PAGE. Steady-state levels of E1B-55K mutants, p53, E2A, and fiber were determined by Western blotting with monoclonal antibodies 2A6, DO-1, B6-6, and a rabbit polyclonal anti-fiber antibody, respectively. Blots were visualized with ECL, as described in Materials and Methods.

    Article Snippet: All antibodies were diluted in the permeabilization and blocking solution. p53 antibody DO-1 (Calbiochem) was diluted 1:100, anti-E1B-55K antibody 9C10 (Calbiochem) was diluted 1:100, and the rabbit anti-E4orf6 antibody 1807-3 was diluted 1:500.

    Techniques: Expressing, Infection, SDS Page, Western Blot

    Coimmunoprecipitation of p53 by E1B-55K mutants. (A and B) Coimmunoprecipitation experiment. A549 cells were either mock infected (mock) or infected with various virus mutants at an MOI of 10. At 24 h postinfection, cells were metabolically labeled with [ 35 S]methionine-cysteine for a 3-h period. Cell extracts were immunoprecipitated with anti-p53 antibody Ab421 (A) or anti-E1B-55K antibody 2A6 (B) and analyzed by SDS-PAGE as described in Materials and Methods. E1B-55K mutant R240A was expressed from ONYX-051, and H260A was expressed from ONYX-053. (C and D) In vitro-translated (and 35 S-labeled) p53 was mixed with lysates prepared from infected A549 cells at 24 h post infection and immunoprecipitated with 2A6 anti-E1B-55K antibody. Immune complexes were separated by SDS-PAGE and visualized either directly by autoradiography (C) or by Western blotting with 2A6 antibody (D).

    Journal: Journal of Virology

    Article Title: Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein

    doi: 10.1128/JVI.75.9.4297-4307.2001

    Figure Lengend Snippet: Coimmunoprecipitation of p53 by E1B-55K mutants. (A and B) Coimmunoprecipitation experiment. A549 cells were either mock infected (mock) or infected with various virus mutants at an MOI of 10. At 24 h postinfection, cells were metabolically labeled with [ 35 S]methionine-cysteine for a 3-h period. Cell extracts were immunoprecipitated with anti-p53 antibody Ab421 (A) or anti-E1B-55K antibody 2A6 (B) and analyzed by SDS-PAGE as described in Materials and Methods. E1B-55K mutant R240A was expressed from ONYX-051, and H260A was expressed from ONYX-053. (C and D) In vitro-translated (and 35 S-labeled) p53 was mixed with lysates prepared from infected A549 cells at 24 h post infection and immunoprecipitated with 2A6 anti-E1B-55K antibody. Immune complexes were separated by SDS-PAGE and visualized either directly by autoradiography (C) or by Western blotting with 2A6 antibody (D).

    Article Snippet: All antibodies were diluted in the permeabilization and blocking solution. p53 antibody DO-1 (Calbiochem) was diluted 1:100, anti-E1B-55K antibody 9C10 (Calbiochem) was diluted 1:100, and the rabbit anti-E4orf6 antibody 1807-3 was diluted 1:500.

    Techniques: Infection, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Mutagenesis, In Vitro, Autoradiography, Western Blot

    Binding of E4orf6 by the E1B-55K mutants. A549 cells were either mock infected (mock) or infected with indicated virus mutants at an MOI of 10. At 24 h postinfection cells were lysed; cleared cell extracts were immunoprecipitated with anti-E1B-55K antibody 2A6. The precipitated materials were separated by SDS-PAGE and analyzed by Western blotting with a rat polyclonal antibody against E4orf6 (A) or with a rat anti-E1B-55K polyclonal antibody (B). (C) The level of E4orf6 expression in the infected cells by straight Western analysis using a polyclonal anti-E4orf6 antibody. dl 355* is a dl 309 derivative lacking the E4orf6 gene.

    Journal: Journal of Virology

    Article Title: Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein

    doi: 10.1128/JVI.75.9.4297-4307.2001

    Figure Lengend Snippet: Binding of E4orf6 by the E1B-55K mutants. A549 cells were either mock infected (mock) or infected with indicated virus mutants at an MOI of 10. At 24 h postinfection cells were lysed; cleared cell extracts were immunoprecipitated with anti-E1B-55K antibody 2A6. The precipitated materials were separated by SDS-PAGE and analyzed by Western blotting with a rat polyclonal antibody against E4orf6 (A) or with a rat anti-E1B-55K polyclonal antibody (B). (C) The level of E4orf6 expression in the infected cells by straight Western analysis using a polyclonal anti-E4orf6 antibody. dl 355* is a dl 309 derivative lacking the E4orf6 gene.

    Article Snippet: All antibodies were diluted in the permeabilization and blocking solution. p53 antibody DO-1 (Calbiochem) was diluted 1:100, anti-E1B-55K antibody 9C10 (Calbiochem) was diluted 1:100, and the rabbit anti-E4orf6 antibody 1807-3 was diluted 1:500.

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

    Indirect immunofluorescent staining of adenovirus- and mock-infected (mock) cells. A549 cells grown on chamber slides were infected with dl 309 or ONYX-015, -051, -052, or -053 at an MOI of 10 or were mock infected. At 24 h postinfection the cells were fixed, permeabilized, and analyzed by indirect immunofluorescent staining using the E1B-55K-specific monoclonal antibody 9C10 (αE1B), p53-specific monoclonal antibody DO-1 (αp53), and E4orf6-specific polyclonal antibody 1807-3 (αE4). Representative fields are shown for all cases.

    Journal: Journal of Virology

    Article Title: Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein

    doi: 10.1128/JVI.75.9.4297-4307.2001

    Figure Lengend Snippet: Indirect immunofluorescent staining of adenovirus- and mock-infected (mock) cells. A549 cells grown on chamber slides were infected with dl 309 or ONYX-015, -051, -052, or -053 at an MOI of 10 or were mock infected. At 24 h postinfection the cells were fixed, permeabilized, and analyzed by indirect immunofluorescent staining using the E1B-55K-specific monoclonal antibody 9C10 (αE1B), p53-specific monoclonal antibody DO-1 (αp53), and E4orf6-specific polyclonal antibody 1807-3 (αE4). Representative fields are shown for all cases.

    Article Snippet: All antibodies were diluted in the permeabilization and blocking solution. p53 antibody DO-1 (Calbiochem) was diluted 1:100, anti-E1B-55K antibody 9C10 (Calbiochem) was diluted 1:100, and the rabbit anti-E4orf6 antibody 1807-3 was diluted 1:500.

    Techniques: Staining, Infection

    Conformational changes in Bak and Bax and Bak-Bax interaction in Ad5 dl 337- and Ad5E1B − -infected HeLa cells. (A) Immunoprecipitation (IP) of Bak from mock-, Ad5 dl 309-, Ad5 dl 337-, and Ad5E1B − -infected cells. Immunoprecipitations were carried out with anti-BRCA-2, anti-Bak(23-37), anti-Bak(Ab-1), and anti-Bak(−TM) antibodies (Abs) from the soluble fraction of cells lysed in CHAPS-containing buffer at 24 and 48 h postinfection. Ad5 dl 309-infected HeLa cell lysate (M) was used as a marker for Bak, Bax, and E1B 19K. Western blotting was carried out on precipitated material with a combination of anti-Bak and anti-Bax antibodies (top four panels) or with anti-E1B 19K antibody (bottom four panels). (B) Immunoprecipitation of Bax from mock-, Ad5 dl 309-, Ad5 dl 337-, and Ad5E1B − -infected cells. Experiment was executed as for panel A except that immunoprecipitation was carried out with anti-Myc, anti-Bax(11-30), anti-Bax(150-165), and anti-Bax(43-61) antibodies. Western blotting was carried out as for panel A.

    Journal: Journal of Virology

    Article Title: Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction

    doi: 10.1128/JVI.76.9.4547-4558.2002

    Figure Lengend Snippet: Conformational changes in Bak and Bax and Bak-Bax interaction in Ad5 dl 337- and Ad5E1B − -infected HeLa cells. (A) Immunoprecipitation (IP) of Bak from mock-, Ad5 dl 309-, Ad5 dl 337-, and Ad5E1B − -infected cells. Immunoprecipitations were carried out with anti-BRCA-2, anti-Bak(23-37), anti-Bak(Ab-1), and anti-Bak(−TM) antibodies (Abs) from the soluble fraction of cells lysed in CHAPS-containing buffer at 24 and 48 h postinfection. Ad5 dl 309-infected HeLa cell lysate (M) was used as a marker for Bak, Bax, and E1B 19K. Western blotting was carried out on precipitated material with a combination of anti-Bak and anti-Bax antibodies (top four panels) or with anti-E1B 19K antibody (bottom four panels). (B) Immunoprecipitation of Bax from mock-, Ad5 dl 309-, Ad5 dl 337-, and Ad5E1B − -infected cells. Experiment was executed as for panel A except that immunoprecipitation was carried out with anti-Myc, anti-Bax(11-30), anti-Bax(150-165), and anti-Bax(43-61) antibodies. Western blotting was carried out as for panel A.

    Article Snippet: Coverslips were stained with either anti-adenovirus 2 goat polyclonal antibody (Accurate Chemical and Scientific, Westbury, N.Y.) diluted 1:60 or anti-Ad5 E1B 55K rat monoclonal antibody (Oncogene Research) diluted 1:60.

    Techniques: Infection, Immunoprecipitation, Marker, Western Blot

    E1B 19K expression during adenovirus infection blocks activation of caspases 9 and 3. Cells were mock, Ad5 dl 309, Ad5 dl 337, or Ad5E1B − infected for 72 h, and representative samples were collected at 0 (for mock only), 12, 24, 36, 48, and 72 h postinfection and analyzed by Western blotting with antibodies against caspase 9, caspase 3, caspase 8, Bid, and PARP. Arrows indicate unprocessed (pro-) and processed forms (where visible) of caspases 9 and 3 and PARP. Lysates of TNF/CHX-treated HeLa cells were located in each panel as controls for caspase processing and substrate cleavage.

    Journal: Journal of Virology

    Article Title: Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction

    doi: 10.1128/JVI.76.9.4547-4558.2002

    Figure Lengend Snippet: E1B 19K expression during adenovirus infection blocks activation of caspases 9 and 3. Cells were mock, Ad5 dl 309, Ad5 dl 337, or Ad5E1B − infected for 72 h, and representative samples were collected at 0 (for mock only), 12, 24, 36, 48, and 72 h postinfection and analyzed by Western blotting with antibodies against caspase 9, caspase 3, caspase 8, Bid, and PARP. Arrows indicate unprocessed (pro-) and processed forms (where visible) of caspases 9 and 3 and PARP. Lysates of TNF/CHX-treated HeLa cells were located in each panel as controls for caspase processing and substrate cleavage.

    Article Snippet: Coverslips were stained with either anti-adenovirus 2 goat polyclonal antibody (Accurate Chemical and Scientific, Westbury, N.Y.) diluted 1:60 or anti-Ad5 E1B 55K rat monoclonal antibody (Oncogene Research) diluted 1:60.

    Techniques: Expressing, Infection, Activation Assay, Western Blot

    Cells deficient in both Bak and Bax are resistant to E1A-induced apoptosis. (A) Bak and Bax deficiency eliminates morphological features of apoptosis. BMK cells that were wild type (W2), deficient in Bax ( bax−/− , or X1), deficient in Bak ( bak−/− , or K1), or deficient in both Bax and Bak ( bax−/− bak−/− , or D2) were either mock infected or infected with Ad5 dl 309, Ad5 dl 337, or Ad5E1B − . At 48 h postinfection, attached and floating cells were examined by phase-contrast microscopy and photographed. (B) Bax and Bak deficiency prevents apoptotic DNA fragmentation induced by E1A during infection with proapoptotic E1B 19K mutant viruses. Low-molecular-weight DNA was isolated in Hirt supernatants from W2, X1, K1, and D2 cells infected as for panel A. (C) Schematic of apoptotic regulation during adenovirus infection. See text for details.

    Journal: Journal of Virology

    Article Title: Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction

    doi: 10.1128/JVI.76.9.4547-4558.2002

    Figure Lengend Snippet: Cells deficient in both Bak and Bax are resistant to E1A-induced apoptosis. (A) Bak and Bax deficiency eliminates morphological features of apoptosis. BMK cells that were wild type (W2), deficient in Bax ( bax−/− , or X1), deficient in Bak ( bak−/− , or K1), or deficient in both Bax and Bak ( bax−/− bak−/− , or D2) were either mock infected or infected with Ad5 dl 309, Ad5 dl 337, or Ad5E1B − . At 48 h postinfection, attached and floating cells were examined by phase-contrast microscopy and photographed. (B) Bax and Bak deficiency prevents apoptotic DNA fragmentation induced by E1A during infection with proapoptotic E1B 19K mutant viruses. Low-molecular-weight DNA was isolated in Hirt supernatants from W2, X1, K1, and D2 cells infected as for panel A. (C) Schematic of apoptotic regulation during adenovirus infection. See text for details.

    Article Snippet: Coverslips were stained with either anti-adenovirus 2 goat polyclonal antibody (Accurate Chemical and Scientific, Westbury, N.Y.) diluted 1:60 or anti-Ad5 E1B 55K rat monoclonal antibody (Oncogene Research) diluted 1:60.

    Techniques: Infection, Microscopy, Mutagenesis, Molecular Weight, Isolation

    E1B 19K prevents the release and degradation of cytochrome c and Smac/DIABLO from mitochondria during adenovirus infection. HeLa cells that were mock, Ad5 dl 309, Ad5 dl 337, or Ad5E1B − infected were harvested at 24 and 48 h postinfection. The total lysate (T) and mitochondrial (M) and cytosolic (C) fractions from each sample were analyzed by Western blotting with antibodies against cytochrome c (top panels), Smac/DIABLO (middle panels), or the mitochondrial marker protein COXII (bottom panels).

    Journal: Journal of Virology

    Article Title: Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction

    doi: 10.1128/JVI.76.9.4547-4558.2002

    Figure Lengend Snippet: E1B 19K prevents the release and degradation of cytochrome c and Smac/DIABLO from mitochondria during adenovirus infection. HeLa cells that were mock, Ad5 dl 309, Ad5 dl 337, or Ad5E1B − infected were harvested at 24 and 48 h postinfection. The total lysate (T) and mitochondrial (M) and cytosolic (C) fractions from each sample were analyzed by Western blotting with antibodies against cytochrome c (top panels), Smac/DIABLO (middle panels), or the mitochondrial marker protein COXII (bottom panels).

    Article Snippet: Coverslips were stained with either anti-adenovirus 2 goat polyclonal antibody (Accurate Chemical and Scientific, Westbury, N.Y.) diluted 1:60 or anti-Ad5 E1B 55K rat monoclonal antibody (Oncogene Research) diluted 1:60.

    Techniques: Infection, Western Blot, Marker

    Deficiency of Bax and Bak enhances the efficiency of viral replication during adenovirus infection. (A) Titers of progeny virus from Ad5 dl 309- or Ad5 dl 337-infected wild-type (W2) or Bax- and Bak-deficient (D2) BMK cells. Infected cells were harvested at 3 and 7 days postinfection. Solid bars indicate titer at 3 days postinfection (P.I.), whereas patterned bars indicate titer at 7 days postinfection. Bars represent the mean titer, and error bars represent the standard variation from the mean of duplicate samples. (B) Expression of E1A, polypeptide III (ppIII), Bax, and Bak in W2, X1, K1, and D2 BMK cells infected with Ad5 dl 309, Ad5 dl 337, or Ad5E1B − . Cells were harvested at 24 and 48 h postinfection and analyzed at the time points indicated by Western blotting with antibodies against E1A (top panels), adenovirus structural proteins (polypeptide III [ppIII], middle panel), and a combination of anti-Bak and anti-Bax antibodies (bottom panel). (C) Indirect immunofluorescence staining for adenovirus E1B 19K in W2 and D2 cells at 1 to 3 days postinfection with Ad5 dl 309 or Ad5 dl 337 or mock infection. Photographs shown are from day 2 postinfection. Asterisk (∗) indicates a sample that was impossible to quantify due to loss of viability. (D) Indirect immunofluorescence staining for adenovirus structural proteins in W2 and D2 cells at 1 to 3 days postinfection with Ad5 dl 309 or Ad5 dl 337 or mock infection. Photographs shown are from day 1 postinfection. Asterisk (∗) indicates a sample that was impossible to quantify due to loss of viability.

    Journal: Journal of Virology

    Article Title: Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction

    doi: 10.1128/JVI.76.9.4547-4558.2002

    Figure Lengend Snippet: Deficiency of Bax and Bak enhances the efficiency of viral replication during adenovirus infection. (A) Titers of progeny virus from Ad5 dl 309- or Ad5 dl 337-infected wild-type (W2) or Bax- and Bak-deficient (D2) BMK cells. Infected cells were harvested at 3 and 7 days postinfection. Solid bars indicate titer at 3 days postinfection (P.I.), whereas patterned bars indicate titer at 7 days postinfection. Bars represent the mean titer, and error bars represent the standard variation from the mean of duplicate samples. (B) Expression of E1A, polypeptide III (ppIII), Bax, and Bak in W2, X1, K1, and D2 BMK cells infected with Ad5 dl 309, Ad5 dl 337, or Ad5E1B − . Cells were harvested at 24 and 48 h postinfection and analyzed at the time points indicated by Western blotting with antibodies against E1A (top panels), adenovirus structural proteins (polypeptide III [ppIII], middle panel), and a combination of anti-Bak and anti-Bax antibodies (bottom panel). (C) Indirect immunofluorescence staining for adenovirus E1B 19K in W2 and D2 cells at 1 to 3 days postinfection with Ad5 dl 309 or Ad5 dl 337 or mock infection. Photographs shown are from day 2 postinfection. Asterisk (∗) indicates a sample that was impossible to quantify due to loss of viability. (D) Indirect immunofluorescence staining for adenovirus structural proteins in W2 and D2 cells at 1 to 3 days postinfection with Ad5 dl 309 or Ad5 dl 337 or mock infection. Photographs shown are from day 1 postinfection. Asterisk (∗) indicates a sample that was impossible to quantify due to loss of viability.

    Article Snippet: Coverslips were stained with either anti-adenovirus 2 goat polyclonal antibody (Accurate Chemical and Scientific, Westbury, N.Y.) diluted 1:60 or anti-Ad5 E1B 55K rat monoclonal antibody (Oncogene Research) diluted 1:60.

    Techniques: Infection, Expressing, Western Blot, Immunofluorescence, Staining