daxx rabbit polyclonal antibody upstate millipore  (Millipore)


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    Name:
    LH Rabbit Polyclonal Antibody
    Description:
    Anti LH is a useful marker in classification of pituitary tumors and the study of pituitary disease It reacts with LH producing cells gonadotrophs
    Catalog Number:
    209a-1
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    Structured Review

    Millipore daxx rabbit polyclonal antibody upstate millipore
    LH Rabbit Polyclonal Antibody
    Anti LH is a useful marker in classification of pituitary tumors and the study of pituitary disease It reacts with LH producing cells gonadotrophs
    https://www.bioz.com/result/daxx rabbit polyclonal antibody upstate millipore/product/Millipore
    Average 99 stars, based on 23606 article reviews
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    daxx rabbit polyclonal antibody upstate millipore - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Proteasome-Dependent Degradation of Daxx by the Viral E1B-55K Protein in Human Adenovirus-Infected Cells ▿"

    Article Title: Proteasome-Dependent Degradation of Daxx by the Viral E1B-55K Protein in Human Adenovirus-Infected Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00074-10

    Reduction of Daxx and PML in shRNA-transduced HAD and HAP cell lines. Cells were fixed and labeled with rabbit polyclonal Daxx antibody (A; HAD cell line) and rabbit polyclonal PML antibody (B; HAP cell line) in combination with Texas Red-conjugated secondary
    Figure Legend Snippet: Reduction of Daxx and PML in shRNA-transduced HAD and HAP cell lines. Cells were fixed and labeled with rabbit polyclonal Daxx antibody (A; HAD cell line) and rabbit polyclonal PML antibody (B; HAP cell line) in combination with Texas Red-conjugated secondary

    Techniques Used: shRNA, Labeling

    2) Product Images from "Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes"

    Article Title: Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt064

    ATRX is reduced via the E1B-55K/E4orf6 E3 ubiquitin ligase complex. ( A ) H1299 control and shCullin5 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154) at moi of 50 FFU per cell. Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx- and Mre11-specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. ( B ) H1299 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154, H5 pm 4139) at moi of 50 FFU per cell. (A) Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx- and Mre11-specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. ( C ) H1299 cells were infected with wild-type (H5 pg 4100) and E1B minus mutant virus (H5 pm 4149) at moi of 50 FFU per cell. Then, 48 h. p. i., total cell extracts were prepared after fractionation of soluble and chromatin fractions as described recently ( 60 ). Proteins were separated by SDS–PAGE and subjected to immunoblotting using Mre11-specific rabbit Ab, Daxx-specific rabbit Ab and ATRX-specific mouse MAb clone 39F, mouse MAb 2A6 (E1B-55K) and mouse MAb RSA3 (E4orf6).
    Figure Legend Snippet: ATRX is reduced via the E1B-55K/E4orf6 E3 ubiquitin ligase complex. ( A ) H1299 control and shCullin5 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154) at moi of 50 FFU per cell. Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx- and Mre11-specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. ( B ) H1299 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154, H5 pm 4139) at moi of 50 FFU per cell. (A) Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx- and Mre11-specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. ( C ) H1299 cells were infected with wild-type (H5 pg 4100) and E1B minus mutant virus (H5 pm 4149) at moi of 50 FFU per cell. Then, 48 h. p. i., total cell extracts were prepared after fractionation of soluble and chromatin fractions as described recently ( 60 ). Proteins were separated by SDS–PAGE and subjected to immunoblotting using Mre11-specific rabbit Ab, Daxx-specific rabbit Ab and ATRX-specific mouse MAb clone 39F, mouse MAb 2A6 (E1B-55K) and mouse MAb RSA3 (E4orf6).

    Techniques Used: Infection, Mutagenesis, SDS Page, Fractionation

    Inhibition of functional Daxx/ATRX complex modulates chromatin structure and condensation. Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. 24 h p.i. ( A ) and 48 h p.i. ( B ), cells were fixed with formaldehyde and analysed by ChIP assays (see ‘Material and Methods’ section). Isolated chromatin was precipitated with histone variant H3.3-specific polyclonal rabbit Ab. The average C t -value was determined from triplicate reactions and normalized with standard curves for each primer pair ( Table 2 ). The y -axis indicates the percentage of immunoprecipitated signal from the input (=100%). Values above 1% of input (dotted line) indicate chromatin/protein binding. The term ‘% of input’ is commonly used as y -axis label for ChIP assays and reflects the percentage of signal normalized to the original sample prior to IP. Values between 0% and 0.05% reflect no binding to promoter sequences. ( C ) Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. Then, 24 h p.i. cells were harvested and total cell extracts were prepared. Proteins were separated by SDS–PAGE and subjected to immunoblotting using Daxx rabbit polyclonal antibody 07–471, ATRX-specific mouse MAb clone 39F, mouse E1B-55K-specific mouse Mab 2A6 and ß-actin mouse MAb AC-15 as a loading control.
    Figure Legend Snippet: Inhibition of functional Daxx/ATRX complex modulates chromatin structure and condensation. Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. 24 h p.i. ( A ) and 48 h p.i. ( B ), cells were fixed with formaldehyde and analysed by ChIP assays (see ‘Material and Methods’ section). Isolated chromatin was precipitated with histone variant H3.3-specific polyclonal rabbit Ab. The average C t -value was determined from triplicate reactions and normalized with standard curves for each primer pair ( Table 2 ). The y -axis indicates the percentage of immunoprecipitated signal from the input (=100%). Values above 1% of input (dotted line) indicate chromatin/protein binding. The term ‘% of input’ is commonly used as y -axis label for ChIP assays and reflects the percentage of signal normalized to the original sample prior to IP. Values between 0% and 0.05% reflect no binding to promoter sequences. ( C ) Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. Then, 24 h p.i. cells were harvested and total cell extracts were prepared. Proteins were separated by SDS–PAGE and subjected to immunoblotting using Daxx rabbit polyclonal antibody 07–471, ATRX-specific mouse MAb clone 39F, mouse E1B-55K-specific mouse Mab 2A6 and ß-actin mouse MAb AC-15 as a loading control.

    Techniques Used: Inhibition, Functional Assay, Infection, Chromatin Immunoprecipitation, Isolation, Variant Assay, Immunoprecipitation, Protein Binding, Binding Assay, SDS Page

    Proteasomal degradation of ATRX in infected and transfected cells. H1299 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154) at moi of 50 FFU per cell. ( A ) Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. Co-immunoprecipitation (IP) of E1B-55K was performed with ATRX-specific mouse MAb clone 39F followed detection of co-precipitated E1B-55K with mouse MAb 2A6. ( B ) Infected cells (as aforementioned) were treated for 6 h with proteasome inhibitor (+MG 132), before total cell extracts were prepared and specific proteins detected as described in A. ( C ) H1299 cells were transfected with pcDNA3-derived plasmids expressing wild-type E1B-55K, E4orf6 or a combination of both. Cells were harvested 48 h. p. i. Total cell extracts were prepared, and specific proteins were immunoprcipitated and detected as described in (A).
    Figure Legend Snippet: Proteasomal degradation of ATRX in infected and transfected cells. H1299 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154) at moi of 50 FFU per cell. ( A ) Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. Co-immunoprecipitation (IP) of E1B-55K was performed with ATRX-specific mouse MAb clone 39F followed detection of co-precipitated E1B-55K with mouse MAb 2A6. ( B ) Infected cells (as aforementioned) were treated for 6 h with proteasome inhibitor (+MG 132), before total cell extracts were prepared and specific proteins detected as described in A. ( C ) H1299 cells were transfected with pcDNA3-derived plasmids expressing wild-type E1B-55K, E4orf6 or a combination of both. Cells were harvested 48 h. p. i. Total cell extracts were prepared, and specific proteins were immunoprcipitated and detected as described in (A).

    Techniques Used: Infection, Transfection, Mutagenesis, SDS Page, Immunoprecipitation, Derivative Assay, Expressing

    3) Product Images from "Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication"

    Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.00402-19

    SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.
    Figure Legend Snippet: SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.

    Techniques Used: Infection, Western Blot

    SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
    Figure Legend Snippet: SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

    Techniques Used: Infection, Western Blot

    SMARCAL1 is phosphorylated during the early stages of Ad infection. (A) A549 cells were either mock infected, treated with MLN4924, or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at 18 h postinfection. Cells were harvested in IP buffer and subjected to immunoprecipitation for SMARCAL1. Anti-SMARCAL1 immunoprecipitates collected on protein G-Sepharose were treated in the absence or presence of λ-phosphatase and then subjected to SDS-PAGE and WB for SMARCAL1. (B) SMARCAL1 was immunoprecipitated from mock-infected and wt Ad5- or wt Ad12-infected A549 cells 18 h postinfection and separated by SDS-PAGE. Protein bands excised from the gel were subjected to trypsinization and mass spectrometric analysis. Identified SMARCAL1 phosphorylated peptides from Ad-infected cells are presented. (C) S123, S129, and S173 are conserved between primates but less well conserved in lower mammals. SMARCAL1 primary sequences from a number of species were aligned using CLUSTAL Omega. Shaded areas indicate conserved residues.
    Figure Legend Snippet: SMARCAL1 is phosphorylated during the early stages of Ad infection. (A) A549 cells were either mock infected, treated with MLN4924, or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at 18 h postinfection. Cells were harvested in IP buffer and subjected to immunoprecipitation for SMARCAL1. Anti-SMARCAL1 immunoprecipitates collected on protein G-Sepharose were treated in the absence or presence of λ-phosphatase and then subjected to SDS-PAGE and WB for SMARCAL1. (B) SMARCAL1 was immunoprecipitated from mock-infected and wt Ad5- or wt Ad12-infected A549 cells 18 h postinfection and separated by SDS-PAGE. Protein bands excised from the gel were subjected to trypsinization and mass spectrometric analysis. Identified SMARCAL1 phosphorylated peptides from Ad-infected cells are presented. (C) S123, S129, and S173 are conserved between primates but less well conserved in lower mammals. SMARCAL1 primary sequences from a number of species were aligned using CLUSTAL Omega. Shaded areas indicate conserved residues.

    Techniques Used: Infection, Immunoprecipitation, SDS Page, Western Blot

    SMARCAL1 is reorganized to viral replication centers during the early stages of Ad infection. A549 cells were either mock infected (i to iii) or infected with 10 PFU/cell of wt Ad5 (iv to vi) or wt Ad12 (vii to ix). At 18 h postinfection, cells were fixed, permeabilized, and costained for SMARCAL1 and RPA2. Arrows indicate regions of RPA2/SMARCAL1 colocalization. In all instances, images were recorded using a Zeiss LSM510-Meta confocal microscope.
    Figure Legend Snippet: SMARCAL1 is reorganized to viral replication centers during the early stages of Ad infection. A549 cells were either mock infected (i to iii) or infected with 10 PFU/cell of wt Ad5 (iv to vi) or wt Ad12 (vii to ix). At 18 h postinfection, cells were fixed, permeabilized, and costained for SMARCAL1 and RPA2. Arrows indicate regions of RPA2/SMARCAL1 colocalization. In all instances, images were recorded using a Zeiss LSM510-Meta confocal microscope.

    Techniques Used: Infection, Microscopy

    ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
    Figure Legend Snippet: ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

    Techniques Used: Infection, Incubation, SDS Page, Western Blot

    4) Product Images from "The E3 Ubiquitin Ligase Activity Associated with the Adenoviral E1B-55K-E4orf6 Complex Does Not Require CRM1-Dependent Export ▿"

    Article Title: The E3 Ubiquitin Ligase Activity Associated with the Adenoviral E1B-55K-E4orf6 Complex Does Not Require CRM1-Dependent Export ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02368-10

    Effects of NES amino acid changes on the stability of the E1B-55K and/or E4orf6 protein and coimmunoprecipitation of E4orf6 with E1B-55K. (A) Amino acid substitutions in E1B-55K and/or E4orf6 mutant viruses. The NES-specific residues in E1B-55K and E4orf6 are indicated by inverted triangles. Numbers refer to amino acid residues in the wt E1B-55K and E4orf6 proteins from H5 pg 4100. Amino acid changes in the E1B and E4orf6 proteins of H5 pm 4101, H5 pm 4116, and H5 pm 4119 are shown. (B) Steady-state expression levels of E1B-55K and E4orf6 proteins. A549 cells were infected with wt and mutant viruses at a multiplicity of 20 FFU per cell. Cells were harvested at the indicated times p.i., and total-cell extracts were prepared. Proteins (20-μg samples for E1B-55K; 100-μg samples for E4orf6) from each time point were separated by SDS-12% PAGE and were subjected to immunoblotting using anti-E1B-55K (α-E1B) mouse MAb 2A6 or anti-E4orf6 mouse MAb RSA3. (C) Coimmunoprecipitation of E4orf6 with E1B-55K. Whole-cell extracts from infected A549 cells were prepared at 16 h p.i., and samples containing 800 μg of protein (or 2 mg for H5 pm 4116 and H5 pm 4119) were coimmunoprecipitated (Ip) with MAb 2A6 and separated by SDS-12% PAGE, followed by immunoblotting with anti-E4orf6 rabbit polyclonal antibody 1807 (lanes 8 to 14). For analysis of steady-state levels of E1B-55K, E4orf6, and β-actin, proteins (20-μg samples for E1B-55K and β-actin; 100-μg samples for E4orf6) were separated by SDS-12% PAGE and were subjected to immunoblotting using anti-E1B-55K mouse MAb 2A6, anti-E4orf6 rabbit polyclonal antibody 1807, or anti-β-actin mouse MAb AC-15 (lanes 1 to 7).
    Figure Legend Snippet: Effects of NES amino acid changes on the stability of the E1B-55K and/or E4orf6 protein and coimmunoprecipitation of E4orf6 with E1B-55K. (A) Amino acid substitutions in E1B-55K and/or E4orf6 mutant viruses. The NES-specific residues in E1B-55K and E4orf6 are indicated by inverted triangles. Numbers refer to amino acid residues in the wt E1B-55K and E4orf6 proteins from H5 pg 4100. Amino acid changes in the E1B and E4orf6 proteins of H5 pm 4101, H5 pm 4116, and H5 pm 4119 are shown. (B) Steady-state expression levels of E1B-55K and E4orf6 proteins. A549 cells were infected with wt and mutant viruses at a multiplicity of 20 FFU per cell. Cells were harvested at the indicated times p.i., and total-cell extracts were prepared. Proteins (20-μg samples for E1B-55K; 100-μg samples for E4orf6) from each time point were separated by SDS-12% PAGE and were subjected to immunoblotting using anti-E1B-55K (α-E1B) mouse MAb 2A6 or anti-E4orf6 mouse MAb RSA3. (C) Coimmunoprecipitation of E4orf6 with E1B-55K. Whole-cell extracts from infected A549 cells were prepared at 16 h p.i., and samples containing 800 μg of protein (or 2 mg for H5 pm 4116 and H5 pm 4119) were coimmunoprecipitated (Ip) with MAb 2A6 and separated by SDS-12% PAGE, followed by immunoblotting with anti-E4orf6 rabbit polyclonal antibody 1807 (lanes 8 to 14). For analysis of steady-state levels of E1B-55K, E4orf6, and β-actin, proteins (20-μg samples for E1B-55K and β-actin; 100-μg samples for E4orf6) were separated by SDS-12% PAGE and were subjected to immunoblotting using anti-E1B-55K mouse MAb 2A6, anti-E4orf6 rabbit polyclonal antibody 1807, or anti-β-actin mouse MAb AC-15 (lanes 1 to 7).

    Techniques Used: Mutagenesis, Expressing, Infection, Polyacrylamide Gel Electrophoresis

    5) Product Images from "Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.9.4547-4558.2002

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

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

    6) Product Images from "Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication"

    Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.00402-19

    SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.
    Figure Legend Snippet: SMARCAL1 is targeted for degradation during Ad infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at the appropriate times postinfection. (A) Ad5 cell lysates were then subjected to WB for SMARCAL1, p53, E1B-55K, E4orf6, and β-actin. (B) Ad12 cell lysates were subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of more than three independent experiments.

    Techniques Used: Infection, Western Blot

    SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
    Figure Legend Snippet: SMARCAL1 is degraded during Ad infection in an E1B-55K/E4orf6- and CRL-dependent manner. (A) A549 cells were either mock infected, infected with wt Ad5, or infected with E1B-55K ( dl 1520), E4orf3 (H5 pm 4150), or E4orf6 (H5 pm 4154) deletion virus. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, E4orf3, E4orf6, and β-actin. (B) A549 cells were either mock infected, infected with wt Ad12, or infected with the E1B-55K ( dl 620) deletion virus. At 24 h and 48 h postinfection, cells were harvested and Western blotted for SMARCAL1, p53, E1B-55K, and β-actin. (C and D) A549 cells were either mock infected or infected with wt Ad5 or wt Ad12 in the absence or presence of 100 nM or 500 nM MLN4924. At 24 h and 48 h postinfection, cells were harvested and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

    Techniques Used: Infection, Western Blot

    SMARCAL1 is phosphorylated during the early stages of Ad infection. (A) A549 cells were either mock infected, treated with MLN4924, or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at 18 h postinfection. Cells were harvested in IP buffer and subjected to immunoprecipitation for SMARCAL1. Anti-SMARCAL1 immunoprecipitates collected on protein G-Sepharose were treated in the absence or presence of λ-phosphatase and then subjected to SDS-PAGE and WB for SMARCAL1. (B) SMARCAL1 was immunoprecipitated from mock-infected and wt Ad5- or wt Ad12-infected A549 cells 18 h postinfection and separated by SDS-PAGE. Protein bands excised from the gel were subjected to trypsinization and mass spectrometric analysis. Identified SMARCAL1 phosphorylated peptides from Ad-infected cells are presented. (C) S123, S129, and S173 are conserved between primates but less well conserved in lower mammals. SMARCAL1 primary sequences from a number of species were aligned using CLUSTAL Omega. Shaded areas indicate conserved residues.
    Figure Legend Snippet: SMARCAL1 is phosphorylated during the early stages of Ad infection. (A) A549 cells were either mock infected, treated with MLN4924, or infected with 10 PFU/cell of wt Ad5 or wt Ad12 and harvested at 18 h postinfection. Cells were harvested in IP buffer and subjected to immunoprecipitation for SMARCAL1. Anti-SMARCAL1 immunoprecipitates collected on protein G-Sepharose were treated in the absence or presence of λ-phosphatase and then subjected to SDS-PAGE and WB for SMARCAL1. (B) SMARCAL1 was immunoprecipitated from mock-infected and wt Ad5- or wt Ad12-infected A549 cells 18 h postinfection and separated by SDS-PAGE. Protein bands excised from the gel were subjected to trypsinization and mass spectrometric analysis. Identified SMARCAL1 phosphorylated peptides from Ad-infected cells are presented. (C) S123, S129, and S173 are conserved between primates but less well conserved in lower mammals. SMARCAL1 primary sequences from a number of species were aligned using CLUSTAL Omega. Shaded areas indicate conserved residues.

    Techniques Used: Infection, Immunoprecipitation, SDS Page, Western Blot

    SMARCAL1 is reorganized to viral replication centers during the early stages of Ad infection. A549 cells were either mock infected (i to iii) or infected with 10 PFU/cell of wt Ad5 (iv to vi) or wt Ad12 (vii to ix). At 18 h postinfection, cells were fixed, permeabilized, and costained for SMARCAL1 and RPA2. Arrows indicate regions of RPA2/SMARCAL1 colocalization. In all instances, images were recorded using a Zeiss LSM510-Meta confocal microscope.
    Figure Legend Snippet: SMARCAL1 is reorganized to viral replication centers during the early stages of Ad infection. A549 cells were either mock infected (i to iii) or infected with 10 PFU/cell of wt Ad5 (iv to vi) or wt Ad12 (vii to ix). At 18 h postinfection, cells were fixed, permeabilized, and costained for SMARCAL1 and RPA2. Arrows indicate regions of RPA2/SMARCAL1 colocalization. In all instances, images were recorded using a Zeiss LSM510-Meta confocal microscope.

    Techniques Used: Infection, Microscopy

    ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.
    Figure Legend Snippet: ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

    Techniques Used: Infection, Incubation, SDS Page, Western Blot

    7) Product Images from "Transforming Potential of the Adenovirus Type 5 E4orf3 Protein"

    Article Title: Transforming Potential of the Adenovirus Type 5 E4orf3 Protein

    Journal: Journal of Virology

    doi:

    Protein analysis of selected AB, ABS, ABT, and ABST cells. (A) The same amount of whole-cell extract was separated on SDS–10% polyacrylamide gels and transferred to a nitrocellulose filter by Western blotting. The same filter was then subsequently probed with anti-E1B-55kDa MAb 2A6, anti-E1A antibody M73, and anti-E1B-19kDa antibody 1G11 followed by enhanced chemiluminescence. Quantitative loading of proteins was determined by probing the filter with the anti-β-actin antibody AC-15. (B) Equal amounts of total cell extracts were subjected to immunoprecipitation using anti-p53 MAb PAb421, anti-E4orf6 MAb RSA3, anti-E4orf3 MAb 6A11, or anti-HA antibody 12CA5. The precipitates were resolved on 10 to 15% protein gels and transferred to nitrocellulose membranes. The p53 and E4orf6 proteins were visualized with antibodies CM-1 and RSA3, respectively, followed by enhanced chemiluminescence. Native E4orf3 and HA-tagged E4orf3 were detected with MAb 6A11 and the HA-specific antibody 12CA5, respectively.
    Figure Legend Snippet: Protein analysis of selected AB, ABS, ABT, and ABST cells. (A) The same amount of whole-cell extract was separated on SDS–10% polyacrylamide gels and transferred to a nitrocellulose filter by Western blotting. The same filter was then subsequently probed with anti-E1B-55kDa MAb 2A6, anti-E1A antibody M73, and anti-E1B-19kDa antibody 1G11 followed by enhanced chemiluminescence. Quantitative loading of proteins was determined by probing the filter with the anti-β-actin antibody AC-15. (B) Equal amounts of total cell extracts were subjected to immunoprecipitation using anti-p53 MAb PAb421, anti-E4orf6 MAb RSA3, anti-E4orf3 MAb 6A11, or anti-HA antibody 12CA5. The precipitates were resolved on 10 to 15% protein gels and transferred to nitrocellulose membranes. The p53 and E4orf6 proteins were visualized with antibodies CM-1 and RSA3, respectively, followed by enhanced chemiluminescence. Native E4orf3 and HA-tagged E4orf3 were detected with MAb 6A11 and the HA-specific antibody 12CA5, respectively.

    Techniques Used: Western Blot, Immunoprecipitation

    8) Product Images from "Transforming Potential of the Adenovirus Type 5 E4orf3 Protein"

    Article Title: Transforming Potential of the Adenovirus Type 5 E4orf3 Protein

    Journal: Journal of Virology

    doi:

    Protein analysis of selected AB, ABS, ABT, and ABST cells. (A) The same amount of whole-cell extract was separated on SDS–10% polyacrylamide gels and transferred to a nitrocellulose filter by Western blotting. The same filter was then subsequently probed with anti-E1B-55kDa MAb 2A6, anti-E1A antibody M73, and anti-E1B-19kDa antibody 1G11 followed by enhanced chemiluminescence. Quantitative loading of proteins was determined by probing the filter with the anti-β-actin antibody AC-15. (B) Equal amounts of total cell extracts were subjected to immunoprecipitation using anti-p53 MAb PAb421, anti-E4orf6 MAb RSA3, anti-E4orf3 MAb 6A11, or anti-HA antibody 12CA5. The precipitates were resolved on 10 to 15% protein gels and transferred to nitrocellulose membranes. The p53 and E4orf6 proteins were visualized with antibodies CM-1 and RSA3, respectively, followed by enhanced chemiluminescence. Native E4orf3 and HA-tagged E4orf3 were detected with MAb 6A11 and the HA-specific antibody 12CA5, respectively.
    Figure Legend Snippet: Protein analysis of selected AB, ABS, ABT, and ABST cells. (A) The same amount of whole-cell extract was separated on SDS–10% polyacrylamide gels and transferred to a nitrocellulose filter by Western blotting. The same filter was then subsequently probed with anti-E1B-55kDa MAb 2A6, anti-E1A antibody M73, and anti-E1B-19kDa antibody 1G11 followed by enhanced chemiluminescence. Quantitative loading of proteins was determined by probing the filter with the anti-β-actin antibody AC-15. (B) Equal amounts of total cell extracts were subjected to immunoprecipitation using anti-p53 MAb PAb421, anti-E4orf6 MAb RSA3, anti-E4orf3 MAb 6A11, or anti-HA antibody 12CA5. The precipitates were resolved on 10 to 15% protein gels and transferred to nitrocellulose membranes. The p53 and E4orf6 proteins were visualized with antibodies CM-1 and RSA3, respectively, followed by enhanced chemiluminescence. Native E4orf3 and HA-tagged E4orf3 were detected with MAb 6A11 and the HA-specific antibody 12CA5, respectively.

    Techniques Used: Western Blot, Immunoprecipitation

    9) Product Images from "Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen"

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

    Journal: The Journal of Clinical Investigation

    doi:

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Infection, Incubation, Flow Cytometry

    10) Product Images from "Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes"

    Article Title: Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt064

    Daxx/ATRX functional complex modulates productive Ad replication. U2OS cells were transfected with either empty vector or pEGFP-ATRX plasmid expressing human ATRX 24 h before infection with wild-type H5 pg 4100 virus or E1B-55K minus virus mutant H5 pm 4149 at moi of 50 FFU per cell. ( A ) U2OS cells were harvested at 48 h. p. i., total cell extracts were prepared and proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and subjected to immunoblotting using mouse ATRX-specific mouse Mab or Daxx-specific rabbit Ab. Corresponding β-actin was included as a loading control. ( B ) Viral particles were harvested 24, 48 and 72 h. p. i., and virus yield was determined by quantitative E2A-72K immunofluorescence staining on HEK293 cells. Averages from three independent experiments are shown. Error bars indicate the standard error of the mean. ( C ) U2OS cells were harvested at 48 h. p. i., total cell extracts were prepared and treated with proteinase K. Quantitative real-time PCR was performed using hexon-specific primers. Ad5 H5 pg 4100 bacmid was used to obtain a standard curve. The results represent the averages from three independent experiments. Error bars indicate the standard error of the mean. ( D ) Total cell extracts were prepared and treated with proteinase K. PCR was performed and identicle amounts of PCR product were separated on an analytic agarose gels (1%) and quantified with Gene Snap Software ( Syngene ).
    Figure Legend Snippet: Daxx/ATRX functional complex modulates productive Ad replication. U2OS cells were transfected with either empty vector or pEGFP-ATRX plasmid expressing human ATRX 24 h before infection with wild-type H5 pg 4100 virus or E1B-55K minus virus mutant H5 pm 4149 at moi of 50 FFU per cell. ( A ) U2OS cells were harvested at 48 h. p. i., total cell extracts were prepared and proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and subjected to immunoblotting using mouse ATRX-specific mouse Mab or Daxx-specific rabbit Ab. Corresponding β-actin was included as a loading control. ( B ) Viral particles were harvested 24, 48 and 72 h. p. i., and virus yield was determined by quantitative E2A-72K immunofluorescence staining on HEK293 cells. Averages from three independent experiments are shown. Error bars indicate the standard error of the mean. ( C ) U2OS cells were harvested at 48 h. p. i., total cell extracts were prepared and treated with proteinase K. Quantitative real-time PCR was performed using hexon-specific primers. Ad5 H5 pg 4100 bacmid was used to obtain a standard curve. The results represent the averages from three independent experiments. Error bars indicate the standard error of the mean. ( D ) Total cell extracts were prepared and treated with proteinase K. PCR was performed and identicle amounts of PCR product were separated on an analytic agarose gels (1%) and quantified with Gene Snap Software ( Syngene ).

    Techniques Used: Functional Assay, Transfection, Plasmid Preparation, Expressing, Infection, Mutagenesis, Polyacrylamide Gel Electrophoresis, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Software

    MNase nuclease accesibility assay to monitor Daxx/ATRX-mediated modulation of chromatin structure. Hepa RG cells were either untreated or infected with H5 pg 4100 Ad5 wild-type at moi of 100 FFU/cell for 24 h. Chromatin sensitivity assays were performed using digestion with 20 U of MNase for the indicated periods followed by an RNase treatment. Digested chromatin was analysed on a 1.4% agarose gel using the G-Box system and Gene-Tools software ( Syngene ). Band intesities were quantified with ImageJ and analysed with GraphPad Prism software. Mono-, di-, tri and poly-nucleosomes are indicated on the right.
    Figure Legend Snippet: MNase nuclease accesibility assay to monitor Daxx/ATRX-mediated modulation of chromatin structure. Hepa RG cells were either untreated or infected with H5 pg 4100 Ad5 wild-type at moi of 100 FFU/cell for 24 h. Chromatin sensitivity assays were performed using digestion with 20 U of MNase for the indicated periods followed by an RNase treatment. Digested chromatin was analysed on a 1.4% agarose gel using the G-Box system and Gene-Tools software ( Syngene ). Band intesities were quantified with ImageJ and analysed with GraphPad Prism software. Mono-, di-, tri and poly-nucleosomes are indicated on the right.

    Techniques Used: Infection, Agarose Gel Electrophoresis, Software

    Daxx/ATRX complex mediates Ad transcriptional repression. ( A ) Hepa RG cells were transfected with luciferase reporter plasmids under Ad promoter control (E1A, E1B, E2early, MLP). Then, 48 h after transfection, samples were lysed, absolute luciferase activity was measured. All samples were normalized for transfection efficiency by measuring Renilla luciferase activity. Promoter activity of E1A, E1B, E2early and MLP promoter in Hep parental cells was normalized to 1. Mean and STD are from three independent experiments. ( B ) HepaRG cells were infected with H5 pg 4100 Ad5 wild-ype at moi of 50 FFU/cell. Then, 24 h. p. i. cells were fixed with formaldehyde and analysed by ChIP assays (see ‘Material and Methods’ section). The average C t -value was determined from triplicate reactions and normalized against non-specific IgG controls with standard curves for each primer pair ( Table 2 ). The y -axis indicates the percentage of immunoprecipitated signal from the input (100%). The white dotted line highlights values > 1% of input, commonly stated as significant chromatin/protein binding.
    Figure Legend Snippet: Daxx/ATRX complex mediates Ad transcriptional repression. ( A ) Hepa RG cells were transfected with luciferase reporter plasmids under Ad promoter control (E1A, E1B, E2early, MLP). Then, 48 h after transfection, samples were lysed, absolute luciferase activity was measured. All samples were normalized for transfection efficiency by measuring Renilla luciferase activity. Promoter activity of E1A, E1B, E2early and MLP promoter in Hep parental cells was normalized to 1. Mean and STD are from three independent experiments. ( B ) HepaRG cells were infected with H5 pg 4100 Ad5 wild-ype at moi of 50 FFU/cell. Then, 24 h. p. i. cells were fixed with formaldehyde and analysed by ChIP assays (see ‘Material and Methods’ section). The average C t -value was determined from triplicate reactions and normalized against non-specific IgG controls with standard curves for each primer pair ( Table 2 ). The y -axis indicates the percentage of immunoprecipitated signal from the input (100%). The white dotted line highlights values > 1% of input, commonly stated as significant chromatin/protein binding.

    Techniques Used: Transfection, Luciferase, Activity Assay, Infection, Chromatin Immunoprecipitation, Immunoprecipitation, Protein Binding

    Daxx/ATRX functional complex modulates productive Ad5 mRNA synthesis. U2OS cells were transfected with either empty vector or pEGFP-ATRX plasmid-expressing human ATRX 24 h before infection with wild-type H5 pg 4100 virus or E1B-55K minus virus mutant H5 pm 4149 at moi of 50 FFU per cell. Then, 24 h. p. i., total RNA was extracted, reverse transcribed and quantified by RT-PCR using primers specific for E1A, E1B-55K, E4orf6 (E4orf6 rev: 5′-CCCTCATAAACACGCTGGAC-3′; E4orf6 fwd: 5′-GCTGGTTTAGGATGGTGGTG-3′) and hexon. Data were normalized to 18S rRNA levels. Values correspond to the mean of triplicates, and error bars indicate the standard error of the mean.
    Figure Legend Snippet: Daxx/ATRX functional complex modulates productive Ad5 mRNA synthesis. U2OS cells were transfected with either empty vector or pEGFP-ATRX plasmid-expressing human ATRX 24 h before infection with wild-type H5 pg 4100 virus or E1B-55K minus virus mutant H5 pm 4149 at moi of 50 FFU per cell. Then, 24 h. p. i., total RNA was extracted, reverse transcribed and quantified by RT-PCR using primers specific for E1A, E1B-55K, E4orf6 (E4orf6 rev: 5′-CCCTCATAAACACGCTGGAC-3′; E4orf6 fwd: 5′-GCTGGTTTAGGATGGTGGTG-3′) and hexon. Data were normalized to 18S rRNA levels. Values correspond to the mean of triplicates, and error bars indicate the standard error of the mean.

    Techniques Used: Functional Assay, Transfection, Plasmid Preparation, Expressing, Infection, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

    ATRX is reduced via the E1B-55K/E4orf6 E3 ubiquitin ligase complex. ( A ) H1299 control and shCullin5 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154) at moi of 50 FFU per cell. Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx- and Mre11-specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. ( B ) H1299 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154, H5 pm 4139) at moi of 50 FFU per cell. (A) Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx- and Mre11-specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. ( C ) H1299 cells were infected with wild-type (H5 pg 4100) and E1B minus mutant virus (H5 pm 4149) at moi of 50 FFU per cell. Then, 48 h. p. i., total cell extracts were prepared after fractionation of soluble and chromatin fractions as described recently ( 60 ). Proteins were separated by SDS–PAGE and subjected to immunoblotting using Mre11-specific rabbit Ab, Daxx-specific rabbit Ab and ATRX-specific mouse MAb clone 39F, mouse MAb 2A6 (E1B-55K) and mouse MAb RSA3 (E4orf6).
    Figure Legend Snippet: ATRX is reduced via the E1B-55K/E4orf6 E3 ubiquitin ligase complex. ( A ) H1299 control and shCullin5 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154) at moi of 50 FFU per cell. Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx- and Mre11-specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. ( B ) H1299 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154, H5 pm 4139) at moi of 50 FFU per cell. (A) Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx- and Mre11-specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. ( C ) H1299 cells were infected with wild-type (H5 pg 4100) and E1B minus mutant virus (H5 pm 4149) at moi of 50 FFU per cell. Then, 48 h. p. i., total cell extracts were prepared after fractionation of soluble and chromatin fractions as described recently ( 60 ). Proteins were separated by SDS–PAGE and subjected to immunoblotting using Mre11-specific rabbit Ab, Daxx-specific rabbit Ab and ATRX-specific mouse MAb clone 39F, mouse MAb 2A6 (E1B-55K) and mouse MAb RSA3 (E4orf6).

    Techniques Used: Infection, Mutagenesis, SDS Page, Fractionation

    Model for Ad5-mediated restriction of cellular Daxx/ATRX chromatin remodelling complexes. ( A ) A schematic representation of known cellular Daxx localizations in human cells. Nuclear Daxx is associated with either PML-NBs or ATRX at heterochromatin foci. Daxx ability to repress transcription is inhibited by its localization to the PML-NB ( 82 ). Cytoplasmic Daxx has been reported to be involved in cell death ( 56 , 83 ). ( B ) Daxx is reported to bind ATRX via two paired amphipathic helices. ATRX acts as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to deacetylation of histone tails (HDAC) and histone variant H3.3 depositioning resulting in transcriptional repression of target promoters. As the Daxx protein has no DNA binding region, ATRX is thought to be the molecular bridge connecting chromatin with Daxx bound to Daxx-interacting sequence-specific transcription factors (yellow sphere). ( C ) Daxx/ATRX repressive complexes assemble on viral genomes. Efficient transcription of Ad5 gene products necessitates inhibiting Daxx repressive complexes and/or preventing their assembly. Binding of E1B-55K triggers Daxx degradation via a proteasome, whereas ATRX restriction additionally requires the E4orf6 protein. These processes displace the repressive Daxx/ATRX complex from the viral genome, relieving negative regulation of Ad transcription.
    Figure Legend Snippet: Model for Ad5-mediated restriction of cellular Daxx/ATRX chromatin remodelling complexes. ( A ) A schematic representation of known cellular Daxx localizations in human cells. Nuclear Daxx is associated with either PML-NBs or ATRX at heterochromatin foci. Daxx ability to repress transcription is inhibited by its localization to the PML-NB ( 82 ). Cytoplasmic Daxx has been reported to be involved in cell death ( 56 , 83 ). ( B ) Daxx is reported to bind ATRX via two paired amphipathic helices. ATRX acts as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to deacetylation of histone tails (HDAC) and histone variant H3.3 depositioning resulting in transcriptional repression of target promoters. As the Daxx protein has no DNA binding region, ATRX is thought to be the molecular bridge connecting chromatin with Daxx bound to Daxx-interacting sequence-specific transcription factors (yellow sphere). ( C ) Daxx/ATRX repressive complexes assemble on viral genomes. Efficient transcription of Ad5 gene products necessitates inhibiting Daxx repressive complexes and/or preventing their assembly. Binding of E1B-55K triggers Daxx degradation via a proteasome, whereas ATRX restriction additionally requires the E4orf6 protein. These processes displace the repressive Daxx/ATRX complex from the viral genome, relieving negative regulation of Ad transcription.

    Techniques Used: Variant Assay, Binding Assay, Sequencing

    Inhibition of functional Daxx/ATRX complex modulates chromatin structure and condensation. Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. 24 h p.i. ( A ) and 48 h p.i. ( B ), cells were fixed with formaldehyde and analysed by ChIP assays (see ‘Material and Methods’ section). Isolated chromatin was precipitated with histone variant H3.3-specific polyclonal rabbit Ab. The average C t -value was determined from triplicate reactions and normalized with standard curves for each primer pair ( Table 2 ). The y -axis indicates the percentage of immunoprecipitated signal from the input (=100%). Values above 1% of input (dotted line) indicate chromatin/protein binding. The term ‘% of input’ is commonly used as y -axis label for ChIP assays and reflects the percentage of signal normalized to the original sample prior to IP. Values between 0% and 0.05% reflect no binding to promoter sequences. ( C ) Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. Then, 24 h p.i. cells were harvested and total cell extracts were prepared. Proteins were separated by SDS–PAGE and subjected to immunoblotting using Daxx rabbit polyclonal antibody 07–471, ATRX-specific mouse MAb clone 39F, mouse E1B-55K-specific mouse Mab 2A6 and ß-actin mouse MAb AC-15 as a loading control.
    Figure Legend Snippet: Inhibition of functional Daxx/ATRX complex modulates chromatin structure and condensation. Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. 24 h p.i. ( A ) and 48 h p.i. ( B ), cells were fixed with formaldehyde and analysed by ChIP assays (see ‘Material and Methods’ section). Isolated chromatin was precipitated with histone variant H3.3-specific polyclonal rabbit Ab. The average C t -value was determined from triplicate reactions and normalized with standard curves for each primer pair ( Table 2 ). The y -axis indicates the percentage of immunoprecipitated signal from the input (=100%). Values above 1% of input (dotted line) indicate chromatin/protein binding. The term ‘% of input’ is commonly used as y -axis label for ChIP assays and reflects the percentage of signal normalized to the original sample prior to IP. Values between 0% and 0.05% reflect no binding to promoter sequences. ( C ) Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. Then, 24 h p.i. cells were harvested and total cell extracts were prepared. Proteins were separated by SDS–PAGE and subjected to immunoblotting using Daxx rabbit polyclonal antibody 07–471, ATRX-specific mouse MAb clone 39F, mouse E1B-55K-specific mouse Mab 2A6 and ß-actin mouse MAb AC-15 as a loading control.

    Techniques Used: Inhibition, Functional Assay, Infection, Chromatin Immunoprecipitation, Isolation, Variant Assay, Immunoprecipitation, Protein Binding, Binding Assay, SDS Page

    Functional inhibition of Daxx/ATRX repressive complex efficiently stimulates Ad5 viral gene expression. Hepa RG cells were infected with wild-type H5 pg 4100 at moi of 50 FFU per cell. ( A ) Viral particles were harvested 24, 48 and 72 h. p. i., and virus yield was determined by quantitative E2A-72K immunofluorescence staining on HEK293 cells. Shown are averages from three independent experiments. Error bars indicate the standard error of the mean. ( B ) Total cell extracts were prepared and treated with proteinase K. PCR was performed, and identicle amounts of PCR product were separated on analytic agarose gels (1%) and quantified with Gene Snap Software ( Syngene ). The results shown represent the averages from two independent experiments.
    Figure Legend Snippet: Functional inhibition of Daxx/ATRX repressive complex efficiently stimulates Ad5 viral gene expression. Hepa RG cells were infected with wild-type H5 pg 4100 at moi of 50 FFU per cell. ( A ) Viral particles were harvested 24, 48 and 72 h. p. i., and virus yield was determined by quantitative E2A-72K immunofluorescence staining on HEK293 cells. Shown are averages from three independent experiments. Error bars indicate the standard error of the mean. ( B ) Total cell extracts were prepared and treated with proteinase K. PCR was performed, and identicle amounts of PCR product were separated on analytic agarose gels (1%) and quantified with Gene Snap Software ( Syngene ). The results shown represent the averages from two independent experiments.

    Techniques Used: Functional Assay, Inhibition, Expressing, Infection, Immunofluorescence, Staining, Polymerase Chain Reaction, Software

    Proteasomal degradation of ATRX in infected and transfected cells. H1299 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154) at moi of 50 FFU per cell. ( A ) Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. Co-immunoprecipitation (IP) of E1B-55K was performed with ATRX-specific mouse MAb clone 39F followed detection of co-precipitated E1B-55K with mouse MAb 2A6. ( B ) Infected cells (as aforementioned) were treated for 6 h with proteasome inhibitor (+MG 132), before total cell extracts were prepared and specific proteins detected as described in A. ( C ) H1299 cells were transfected with pcDNA3-derived plasmids expressing wild-type E1B-55K, E4orf6 or a combination of both. Cells were harvested 48 h. p. i. Total cell extracts were prepared, and specific proteins were immunoprcipitated and detected as described in (A).
    Figure Legend Snippet: Proteasomal degradation of ATRX in infected and transfected cells. H1299 cells were infected with wild-type (H5 pg 4100) and mutant viruses (H5 pm 4149, H5 pm 4154) at moi of 50 FFU per cell. ( A ) Total cell extracts were prepared 48 h. p. i., and proteins were separated by SDS–PAGE and subjected to immunoblotting using mouse MAb 2A6 (E1B-55K), mouse MAb RSA3 (E4orf6), Daxx specific rabbit Ab and ATRX-specific mouse MAb clone 39F. β-actin was included as a loading control. Co-immunoprecipitation (IP) of E1B-55K was performed with ATRX-specific mouse MAb clone 39F followed detection of co-precipitated E1B-55K with mouse MAb 2A6. ( B ) Infected cells (as aforementioned) were treated for 6 h with proteasome inhibitor (+MG 132), before total cell extracts were prepared and specific proteins detected as described in A. ( C ) H1299 cells were transfected with pcDNA3-derived plasmids expressing wild-type E1B-55K, E4orf6 or a combination of both. Cells were harvested 48 h. p. i. Total cell extracts were prepared, and specific proteins were immunoprcipitated and detected as described in (A).

    Techniques Used: Infection, Transfection, Mutagenesis, SDS Page, Immunoprecipitation, Derivative Assay, Expressing

    Daxx/ATRX complex represses Ad5 mRNA synthesis. HepaRG cells were infected with H5 pg 4100 Ad5 wild-type at moi of 50 FFU/cell. Then, 24 h. p. i., total RNA was extracted, reverse transcribed and quantified by RT-PCR using primers specific for E1A (E1A fwd: 5′-GTGCCCCATTAAACCAGTTG-3′; E1A rev: 5′-GGCGTTTACAGCTCAAGTCC-3′), E1B-55K (E1B fwd: 5′-GAGGGTAACTCCAGGG TGCG-3′; E1B rev: 5′-TTTCACTAGCATGAaGCAACCACA-3′, E2A/DBP and hexon (hexon rev: 5′-GAACGGTGTGCGCAGGTA-3′; hexon fwd 5′-CGCTGGACATGACTTTTG AG-3′. Data were normalized to 18S rRNA levels (18S rRNA fwd: 5′-cggctaccacatccaaggaa-3′; 18S rRNA rev: 5′-GCTGGAATTACCGCGGCT-3′). Values correspond to the mean of triplicates, and error bars indicate the standard error of the mean.
    Figure Legend Snippet: Daxx/ATRX complex represses Ad5 mRNA synthesis. HepaRG cells were infected with H5 pg 4100 Ad5 wild-type at moi of 50 FFU/cell. Then, 24 h. p. i., total RNA was extracted, reverse transcribed and quantified by RT-PCR using primers specific for E1A (E1A fwd: 5′-GTGCCCCATTAAACCAGTTG-3′; E1A rev: 5′-GGCGTTTACAGCTCAAGTCC-3′), E1B-55K (E1B fwd: 5′-GAGGGTAACTCCAGGG TGCG-3′; E1B rev: 5′-TTTCACTAGCATGAaGCAACCACA-3′, E2A/DBP and hexon (hexon rev: 5′-GAACGGTGTGCGCAGGTA-3′; hexon fwd 5′-CGCTGGACATGACTTTTG AG-3′. Data were normalized to 18S rRNA levels (18S rRNA fwd: 5′-cggctaccacatccaaggaa-3′; 18S rRNA rev: 5′-GCTGGAATTACCGCGGCT-3′). Values correspond to the mean of triplicates, and error bars indicate the standard error of the mean.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction

    11) Product Images from "The Combination of a Low-Dose Chemotherapeutic Agent, 5-Fluorouracil, and an Adenoviral Tumor Vaccine Has a Synergistic Benefit on Survival in a Tumor Model System"

    Article Title: The Combination of a Low-Dose Chemotherapeutic Agent, 5-Fluorouracil, and an Adenoviral Tumor Vaccine Has a Synergistic Benefit on Survival in a Tumor Model System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067904

    Effect of depleting MDSCs from variously vaccinated tumor-bearing mice on survival and tumor-specific T lymphocyte responses. A . Tumor-bearing mice were treated with multiple i.p. doses of RB6-8C5 (as described in materials and methods section) and draining lymph node and spleen cells were isolated after 6 days of depletion (day 12 post-tumor challenge) and stained for the presence of gMDSCs (CD11b + Ly6C +low Ly6G + ) and mMDSCs (CD11b + Ly6C +hi Ly6G − ). Data is presented as a percentage of total lymph node or spleen populations. Student T-test was used to determine statistical significance. B. Survival analysis: C57BL/6 mice were challenged with E.G7 and then, 7 days post tumor challenge, were treated as indicated. Survival curve represents pooled data from 2 independent experiments where a total of n = 8 mice/treatment group were used. Statistical analysis (Log-rank (Mantel-Cox) test) of survival data revealed that only the Ad5-OVA plus 5-FU treatment to be significantly different from all other treatments (*** P
    Figure Legend Snippet: Effect of depleting MDSCs from variously vaccinated tumor-bearing mice on survival and tumor-specific T lymphocyte responses. A . Tumor-bearing mice were treated with multiple i.p. doses of RB6-8C5 (as described in materials and methods section) and draining lymph node and spleen cells were isolated after 6 days of depletion (day 12 post-tumor challenge) and stained for the presence of gMDSCs (CD11b + Ly6C +low Ly6G + ) and mMDSCs (CD11b + Ly6C +hi Ly6G − ). Data is presented as a percentage of total lymph node or spleen populations. Student T-test was used to determine statistical significance. B. Survival analysis: C57BL/6 mice were challenged with E.G7 and then, 7 days post tumor challenge, were treated as indicated. Survival curve represents pooled data from 2 independent experiments where a total of n = 8 mice/treatment group were used. Statistical analysis (Log-rank (Mantel-Cox) test) of survival data revealed that only the Ad5-OVA plus 5-FU treatment to be significantly different from all other treatments (*** P

    Techniques Used: Mouse Assay, Isolation, Staining

    Anti-tumor effect and survival in mice treated with Ad5-OVA and/or 5-FU. C57BL/6 mice were challenged with E.G7 and then, 7 days post tumor challenge, given: no treatment (naïve); Ad5-OVA; Ad5-OVA +5-FU; or 5-FU. A–D. The tumor volumes for each mouse from one representative experiment are shown. E. Survival graph representing four pooled experiments. Total number of mice for each treatment was: n = 15 for naïve group, n = 14 for Ad5-OVA alone group, n = 21 for Ad5-OVA +5-FU group, n = 10 for Ad5-LacZ +5-FU group, and n = 15 for the 5-FU alone group. Statistical analysis (Log-rank (Mantel-Cox) test) of survival data revealed that mice survived significantly longer, compared to untreated mice, when treated with Ad5-OVA alone (p
    Figure Legend Snippet: Anti-tumor effect and survival in mice treated with Ad5-OVA and/or 5-FU. C57BL/6 mice were challenged with E.G7 and then, 7 days post tumor challenge, given: no treatment (naïve); Ad5-OVA; Ad5-OVA +5-FU; or 5-FU. A–D. The tumor volumes for each mouse from one representative experiment are shown. E. Survival graph representing four pooled experiments. Total number of mice for each treatment was: n = 15 for naïve group, n = 14 for Ad5-OVA alone group, n = 21 for Ad5-OVA +5-FU group, n = 10 for Ad5-LacZ +5-FU group, and n = 15 for the 5-FU alone group. Statistical analysis (Log-rank (Mantel-Cox) test) of survival data revealed that mice survived significantly longer, compared to untreated mice, when treated with Ad5-OVA alone (p

    Techniques Used: Mouse Assay

    12) Product Images from "Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes"

    Article Title: Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt064

    Inhibition of functional Daxx/ATRX complex modulates chromatin structure and condensation. Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. 24 h p.i. ( A ) and 48 h p.i. ( B ), cells were fixed with formaldehyde and analysed by ChIP assays (see ‘Material and Methods’ section). Isolated chromatin was precipitated with histone variant H3.3-specific polyclonal rabbit Ab. The average C t -value was determined from triplicate reactions and normalized with standard curves for each primer pair ( Table 2 ). The y -axis indicates the percentage of immunoprecipitated signal from the input (=100%). Values above 1% of input (dotted line) indicate chromatin/protein binding. The term ‘% of input’ is commonly used as y -axis label for ChIP assays and reflects the percentage of signal normalized to the original sample prior to IP. Values between 0% and 0.05% reflect no binding to promoter sequences. ( C ) Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. Then, 24 h p.i. cells were harvested and total cell extracts were prepared. Proteins were separated by SDS–PAGE and subjected to immunoblotting using Daxx rabbit polyclonal antibody 07–471, ATRX-specific mouse MAb clone 39F, mouse E1B-55K-specific mouse Mab 2A6 and ß-actin mouse MAb AC-15 as a loading control.
    Figure Legend Snippet: Inhibition of functional Daxx/ATRX complex modulates chromatin structure and condensation. Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. 24 h p.i. ( A ) and 48 h p.i. ( B ), cells were fixed with formaldehyde and analysed by ChIP assays (see ‘Material and Methods’ section). Isolated chromatin was precipitated with histone variant H3.3-specific polyclonal rabbit Ab. The average C t -value was determined from triplicate reactions and normalized with standard curves for each primer pair ( Table 2 ). The y -axis indicates the percentage of immunoprecipitated signal from the input (=100%). Values above 1% of input (dotted line) indicate chromatin/protein binding. The term ‘% of input’ is commonly used as y -axis label for ChIP assays and reflects the percentage of signal normalized to the original sample prior to IP. Values between 0% and 0.05% reflect no binding to promoter sequences. ( C ) Hepa RG cells were infected with H5pg4100 Ad5 wild-type at moi of 5 and 100 FFU/cell. Then, 24 h p.i. cells were harvested and total cell extracts were prepared. Proteins were separated by SDS–PAGE and subjected to immunoblotting using Daxx rabbit polyclonal antibody 07–471, ATRX-specific mouse MAb clone 39F, mouse E1B-55K-specific mouse Mab 2A6 and ß-actin mouse MAb AC-15 as a loading control.

    Techniques Used: Inhibition, Functional Assay, Infection, Chromatin Immunoprecipitation, Isolation, Variant Assay, Immunoprecipitation, Protein Binding, Binding Assay, SDS Page

    13) Product Images from "Suppression of Nicotine-Induced Pathophysiology by an Adenovirus Hexon-Based Antinicotine Vaccine"

    Article Title: Suppression of Nicotine-Induced Pathophysiology by an Adenovirus Hexon-Based Antinicotine Vaccine

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2012.245

    Production of HexonAM1 and experimental design. (A) Production of HexonAM1. Shown is a schematic of conjugation of a nicotine analog (AM1) to the purified hexon of disrupted E1 − E3 − Ad5. (B) Experimental design with timing of presensitization
    Figure Legend Snippet: Production of HexonAM1 and experimental design. (A) Production of HexonAM1. Shown is a schematic of conjugation of a nicotine analog (AM1) to the purified hexon of disrupted E1 − E3 − Ad5. (B) Experimental design with timing of presensitization

    Techniques Used: Conjugation Assay, Purification

    14) Product Images from "Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection"

    Article Title: Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

    Journal: Cell Cycle

    doi: 10.4161/cc.11.3.19052

    Absence of Mdm2 permits efficient HAdV gene expression in mouse embryonic fibroblasts (MEFs). MEFs of the indicated genotypes (p53 KO, Mdm2/p53 or Mdm4/p53 double knockout, DKO) were uninfected or infected with the indicated viruses (MOI of 10). The infected
    Figure Legend Snippet: Absence of Mdm2 permits efficient HAdV gene expression in mouse embryonic fibroblasts (MEFs). MEFs of the indicated genotypes (p53 KO, Mdm2/p53 or Mdm4/p53 double knockout, DKO) were uninfected or infected with the indicated viruses (MOI of 10). The infected

    Techniques Used: Expressing, Double Knockout, Infection

    Depletion of Mdm2 or Mdm4 enhances HAdV gene expression. Lentiviral vector carrying a control, Mdm2 or Mdm4 shRNA was stably transduced in HCT116. The cells were uninfected (mock) or infected with the indicated MOIs of wt Ad5 or Ad12. Cells were harvested
    Figure Legend Snippet: Depletion of Mdm2 or Mdm4 enhances HAdV gene expression. Lentiviral vector carrying a control, Mdm2 or Mdm4 shRNA was stably transduced in HCT116. The cells were uninfected (mock) or infected with the indicated MOIs of wt Ad5 or Ad12. Cells were harvested

    Techniques Used: Expressing, Plasmid Preparation, shRNA, Stable Transfection, Infection

    HAdV infection downregulates Mdm2 and Mdm4. (A) A549 cells were uninfected (mock) or infected (at the MOI of 10 for each) with wt Ad5, E1B-55K-deleted Ad5 mutant ( dl 1520, also known as ONYX-015), wt Ad12, replication-deficient Ad5 expressing GFP fusion
    Figure Legend Snippet: HAdV infection downregulates Mdm2 and Mdm4. (A) A549 cells were uninfected (mock) or infected (at the MOI of 10 for each) with wt Ad5, E1B-55K-deleted Ad5 mutant ( dl 1520, also known as ONYX-015), wt Ad12, replication-deficient Ad5 expressing GFP fusion

    Techniques Used: Infection, Mutagenesis, Expressing

    HAdV-mediated downregulation of Mdm2 and Mdm4 partially depends on proteasome but not Cul5. (A) SASJ-1 cells were uninfected (mock) or infected with the indicated viruses (MOI of 10). Cells were harvested 48 hpi for western blotting analysis with the
    Figure Legend Snippet: HAdV-mediated downregulation of Mdm2 and Mdm4 partially depends on proteasome but not Cul5. (A) SASJ-1 cells were uninfected (mock) or infected with the indicated viruses (MOI of 10). Cells were harvested 48 hpi for western blotting analysis with the

    Techniques Used: Infection, Western Blot

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    Immunohistochemistry:

    Article Title: Reversible pathologic and cognitive phenotypes in an inducible model of Alzheimer-amyloidosis
    Article Snippet: .. In immunohistochemistry, we used monoclonal antibodies 6E10 and rabbit polyclonal antibody OC (EMD Millipore, Billerica, MA) ( )], which recognize the human Aβ sequence and fibrillar Aβ, respectively. .. For ELISA analysis, we used human anti-Aβ42 (Rabbit Monoclonal Clone 1-11-3, Covance, Princeton, NJ) and mAb 4G8.

    Incubation:

    Article Title: Deacetylation of p53 induces autophagy by suppressing Bmf expression
    Article Snippet: .. Sonicated nuclear fractions were incubated with mouse IgG1 monoclonal antibody to p53 (sc-98 Pab1801; Santa Cruz Biotechnology, Inc.), rabbit polyclonal antibody to HDAC1 (06–720; EMD Millipore), rabbit polyclonal antibody to acetyl-H4 (06–598; EMD Millipore), rabbit polyclonal antibody to acetyl-H3 (06-599B; EMD Millipore), mouse IgG1, or rabbit as a control (CBL600; EMD Millipore). .. DNA identification was confirmed with PCR using the following primers specific for the Bmf promoter: region −560, 5′-ACCTAAGGGCTCCCCTGGA-3′ and 5′-GCAGGTCGGAAGAAAACTGCAGC-3′, and region −97, 5′-TTGGCGCTTCACTCGCCATT-3′ and 5′-ATCCCGCAAACAGCTGAT-3′.

    Article Title: Augmentation of Kv4.2-encoded Currents by Accessory Dipeptidyl Peptidase 6 and 10 Subunits Reflects Selective Cell Surface Kv4.2 Protein Stabilization
    Article Snippet: .. The membranes were then incubated with a mouse monoclonal anti-Kv4.2 or anti-KChIP3 antibody or with a rabbit polyclonal anti-GFP (Millipore, Billerica, MA) or anti-DPP6 ( ) antibody at 4 °C overnight. .. The anti-Kv4.2 and anti-KChIP3 antibodies were developed by and obtained from the UC Davis/National Institutes of Health NeuroMab facility, supported by National Institutes of Health Grant U24NS050606 and maintained by the University of California at Davis; the anti-DPP6 antibody was obtained from Dr. Bernardo Rudy.

    Generated:

    Article Title: Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation
    Article Snippet: .. Rabbit polyclonal antibody to p21 (Cat# PC55), generated against a recombinant protein consisting amino acids 15–61 of mouse p21, was purchased from Oncogene Research Products (Cambridge, MA). .. A monoclonal antibody for p27, obtained from mouse ascites by epitope-affinity chromatography using full-length human recombinant p27 protein, was obtained from Zymed Laboratories Inc. (San Francisco, CA).

    Sequencing:

    Article Title: Reversible pathologic and cognitive phenotypes in an inducible model of Alzheimer-amyloidosis
    Article Snippet: .. In immunohistochemistry, we used monoclonal antibodies 6E10 and rabbit polyclonal antibody OC (EMD Millipore, Billerica, MA) ( )], which recognize the human Aβ sequence and fibrillar Aβ, respectively. .. For ELISA analysis, we used human anti-Aβ42 (Rabbit Monoclonal Clone 1-11-3, Covance, Princeton, NJ) and mAb 4G8.

    Sonication:

    Article Title: Deacetylation of p53 induces autophagy by suppressing Bmf expression
    Article Snippet: .. Sonicated nuclear fractions were incubated with mouse IgG1 monoclonal antibody to p53 (sc-98 Pab1801; Santa Cruz Biotechnology, Inc.), rabbit polyclonal antibody to HDAC1 (06–720; EMD Millipore), rabbit polyclonal antibody to acetyl-H4 (06–598; EMD Millipore), rabbit polyclonal antibody to acetyl-H3 (06-599B; EMD Millipore), mouse IgG1, or rabbit as a control (CBL600; EMD Millipore). .. DNA identification was confirmed with PCR using the following primers specific for the Bmf promoter: region −560, 5′-ACCTAAGGGCTCCCCTGGA-3′ and 5′-GCAGGTCGGAAGAAAACTGCAGC-3′, and region −97, 5′-TTGGCGCTTCACTCGCCATT-3′ and 5′-ATCCCGCAAACAGCTGAT-3′.

    Recombinant:

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    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
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    anti ad5 e1b 55k rat monoclonal antibody - by Bioz Stars, 2020-09
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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 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ad5 e1b/product/Millipore
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    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

    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