anti thbs1  (Bio-Techne corporation)

Journal: Cardiovascular Research

Article Title: Doxorubicin-induced cardiovascular toxicity: a longitudinal evaluation of functional and molecular markers

doi: 10.1093/cvr/cvad136

Figure Lengend Snippet: Proteomic analysis of aortic samples from DOX-treated mice at Weeks 2 and 6 and validation of two candidate biomarkers with western blotting. Plots of differentially expressed proteins at Weeks 2 and 6 ( A ). For A , horizontal grey line indicates significance threshold ( P < 0.05); up- and down-regulated proteins are indicated in blue and red, respectively; differentially expressed proteins are abbreviated (full names are listed in abbreviations list). Western blotting shows and corroborates higher levels of SERPINA3 in DOX-treated mice at Weeks 2 and 6, which persist at Week 9 ( B ). Western blotting also confirms higher levels of THBS1 in the DOX-treated group at Weeks 2 and 6 and shows a trend ( P = 0.052) towards increased THBS1 levels at Week 9 ( B ). Representative western blot images for SERPINA3 and THBS1 at Weeks 2, 6, and 9 ( C ). For C , ‘V’ and ‘D’ indicate lanes with vehicle and DOX aortic samples, respectively. D* on the blot at Week 9 contains a DOX sample for which no protein was detected and was excluded from analysis as such. For A , n = 8 in vehicle and DOX group for both Week 2 and Week 6. For B , vehicle group: n = 5, n = 6, and n = 6 at Weeks 2, 6, and 9, respectively; DOX group: n = 5, n = 6, and n = 5 at Weeks 2, 6, and 9, respectively. For B , Mann–Whitney U test for each time point. * and ** P < 0.05 and 0.01. Abbreviations for differentially expressed proteins in proteomics: SERPINA1E, α-1-antitrypsin; HMGN2, Non-histone chromosomal protein HMG-17; SERPINA3N, α-1-antichymotrypsin orthologue n; MT2, Metallothionein-2; MBL2, Mannose-binding protein C; MT1, Metallothionein-1; THBS1, Thrombospondin-1; FTL1, Ferritin light chain 1; COL14A1, Collagen α-1 (XIV) chain; LGALS3BP, Galectin-3-binding protein; RPL26, 60S ribosomal protein L26; IGHM, Immunoglobulin heavy constant µ; HAMP, Hepcidin; APOA2, Apolipoprotein A-II; HBA, Hemoglobin subunit α; HBB-B1, Hemoglobin subunit β-1; CA1, Carbonic anhydrase 1; KRT15, Keratin (type I cytoskeletal 15); SPTA1, Spectrin α; CA2; Carbonic anhydrase 2; RPL6, 60S ribosomal protein L6; VWA1, von Willebrand factor A domain-containing protein 1; RPL37A, 60S ribosomal protein L37a; CAPG, Macrophage-capping protein; RAD23A, UV excision repair protein RAD23 homolog A; IGHG1, Ig γ-1 chain C region (membrane-bound form); SLC4A1, Band 3 anion transport protein; ACLY, ATP-citrate synthase; ATPA1, Sodium/potassium-transporting ATPase subunit alpha-1.

Article Snippet: For validation of anti-THBS1 and anti-SERPINA3 primary antibodies, recombinant mouse THBS1 (catalogue number 7859-TH-050) and SERPINA3 (catalogue number 4709-PI-010) proteins were used as positive controls, which were purchased from Bio-Techne (Dublin, Ireland).

Techniques: Western Blot, MANN-WHITNEY, Binding Assay, Membrane

Journal: Cardiovascular Research

Article Title: Doxorubicin-induced cardiovascular toxicity: a longitudinal evaluation of functional and molecular markers

doi: 10.1093/cvr/cvad136

Figure Lengend Snippet: Quantification of SERPINA3 and THBS1 levels in plasma of control and AICT patients using ELISA. Schematic overview of the patient cohorts ( A ). AICT patients exhibited higher SERPINA3 and THBS1 levels compared with the control group ( B ). Both SERPINA3 and THBS1 showed an inverse Pearson correlation with LVEF ( C ). For B , data are presented as Tukey box plots with median (horizontal line) and the 25th and 75th percentile whiskers. For C , white and red dots represent control and AICT patients, respectively. n numbers are shown in A . For B , Mann–Whitney U test. * and **** P < 0.05 and 0.0001.

Article Snippet: For validation of anti-THBS1 and anti-SERPINA3 primary antibodies, recombinant mouse THBS1 (catalogue number 7859-TH-050) and SERPINA3 (catalogue number 4709-PI-010) proteins were used as positive controls, which were purchased from Bio-Techne (Dublin, Ireland).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled telomeric dsDNA and binding was assessed by EMSA. Excess of unlabeled telomeric and non-telomeric dsDNA were added as competitors. Samples were migrated on non-denaturing acrylamide gel, dried and exposed to X-ray films. B) Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled non-telomeric dsDNA and binding was assessed by EMSA. C) Recombinant MBP and MBP-TRF2 were coated to the wells of a 96 well-plate and incubated with HaeIII digested DIG-labeled HHV-6A DNA (25 ng/condition) in the presence or absence of competitors. After washing, bound DNA was quantified by adding peroxidase-labeled anti-DIG antibodies and substrate. Results are expressed as mean absorbance +SD of triplicate values. Experiment is representative of two additional experiments. * P<0.001.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Recombinant, Incubation, Labeling, Binding Assay, Acrylamide Gel Assay

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) Schematic representation of the HHV-6A/B genome. The DR6 probe used for hybridization is shown in red. Uninfected and HHV-6A-infected HSB-2 cells (B-D) or uninfected and HHV-6B-infectd Molt-3 cells (E-F) were analyzed for TRF2 binding to viral DNA using ChIP. The input was hybridized with Alu probe to assess quantity of starting material. Anti-IgG (negative control), anti-PolII (positive control) or TRF2 antibodies were used for immunoprecipitation. B) QPCR detection of GAPDH DNA following ChIP. Results are expressed as fold increase over control IgG. C and E) Eluted DNA was hybridized with 32 P-labeled Alu, telomeric (TTAGGG) 3 or HHV-6A (DR6) probes. After hybridization the membranes were washed and exposed to X-ray films. D and F) Densitometric analysis of relative binding of TRF2 to telomeric and viral DNA. Results of one experiment representative of three are presented and are expressed as signal after normalization to input.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Hybridization, Infection, Binding Assay, Negative Control, Positive Control, Immunoprecipitation, Control, Labeling

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: U2OS cells were infected with HHV-6A and analyzed for TRF2 and IE2 expression at 24h, 48h and 72h post-infection by dual color immunofluorescence. A) Representative immunofluorescence of TRF2 and IE2 expression in bystander and IE2 expressing cells at 24, 48h and 72h post infection. B) Mean relative TRF2 expression ± SD in uninfected (blue), IE2- (green-uninfected bystander) or IE2+ (red-infected) cells at 24h, 48h and 72h post infection. Each symbol represents the relative TRF2 expression from a single nucleus.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Infection, Expressing, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) U2OS cells were mock-infected or infected with HHV-6A for 48h after which cells were processed for IF-FISH. Telomeres were labeled in magenta, TRF2 in green and IE2 in red. The panels in the middle row show images of cells with IE2 patches overlapping with large, diffuse TRF2 and telomeric staining (rectangles). The panels in the third row represent infected cells with punctate IE2 pattern colocalizing with TRF2 and telomeres (dashed squares). The colocalization of IE2, TRF2 and telomeres are shown in both 2D and 3D images. B) Uninfected and HHV-6A-infected U2OS cells were transfected with an empty vector, a myc-tagged-TRF1 expression vector. Forty-eight hours later cells were processed for IF-FISH. TRF1 was labeled in green and IE2 in red. Nuclei were stained with DAPI. Images on the far right show 2D colocalization of TRF1 with IE2.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Infection, Labeling, Staining, Transfection, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) A stick diagram of the IE2 protein with various domains identified is presented. B) Colocalization of HHV-6A IE2 protein with telomeres in the absence of viral DNA. U2OS cells were transfected with an empty vector, with IE2 expression vector or with IE2 Δ1290–1500 expression vector. Forty-eight hours later cells were processed for dual color immunofluorescence. Telomeres were labeled in cyan and IE2 in red. Nuclei are outlined by dashed lines. Examples of IE2 colocalizing with telomeres are presented in a 3D view (white arrows). C) The graph represents the mean ± SD % of WT IE2 and Δ1290–1500 IE2 localizing with telomeres. D) Lack of colocalization between HHV-6A p41 and telomeres in uninfected cells. U2OS cells were transfected with an empty vector or with a p41 expression vector. Forty-eight hours later cells were processed for dual color immunofluorescence. TRF2 was labeled green, p41 in red and nuclei outlined by a dashed line.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Labeling

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: U2OS cells were transduced with a lentiviral vector coding for a Dox inducible control shRNA (shCtrl) or a shRNA against TRF2 (shTRF2) and selected with puromycin +/- Dox for a week. A) Western blot analysis of TRF2 expression one week post selection. Membranes were also probed with anti-tubulin antibodies to show the input material loaded. B) One week post selection, +Dox cells were infected with HHV-6A for 48h and processed for IFA using anti-TRF2 (green) and anti-IE2 (red). Cells with IE2 in punctate form and cells with large patchy IE2, likely to represent VRC, are shown. Nuclei are outlined by dashed lines. C) The percentage of HHV-6A infected cells (from B) was estimated after counting a minimum of 700 cells and scoring the IE2 + ones. Results are expressed as mean %IE2 + cells ± SD. D) Mean percentage ± SD of IE2 localizing with telomeres in the presence (shCtrl +Dox) and absence (shTRF2 +Dox) of TRF2. Each dot represents the % of IE2 foci localizing with telomeres in one nucleus. ****p<0.0001. E) IF-FISH confocal images of shCtrl (+Dox) and shTRF2 (+Dox) cells analyzed for TRF2 (green), IE2 (red) and telomeres (cyan). Nuclei are outlined by dashed circles. Examples of IE2 localizing with telomeres (top row) or not found with telomeres (bottom row) are highlighted by the dashed polygons.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Transduction, Plasmid Preparation, Control, shRNA, Western Blot, Expressing, Selection, Infection

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) U2OS cells were transduced with lentiviral vectors expressing a scrambles shRNA (shCtrl) or a shTRF2. After a week of selection, cells were monitored for TRF2 expression by western blot. B) After a week of selection, shCtrl and shTRF2 treated cells were infected with HHV-6A or HHV-6B. After 30 days, DNA was isolated and the relative frequency of integration, relative to shCtrl set at 100%, estimated by ddPCR. *p<0.05.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Transduction, Expressing, shRNA, Selection, Western Blot, Infection, Isolation

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled telomeric dsDNA and binding was assessed by EMSA. Excess of unlabeled telomeric and non-telomeric dsDNA were added as competitors. Samples were migrated on non-denaturing acrylamide gel, dried and exposed to X-ray films. B) Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled non-telomeric dsDNA and binding was assessed by EMSA. C) Recombinant MBP and MBP-TRF2 were coated to the wells of a 96 well-plate and incubated with HaeIII digested DIG-labeled HHV-6A DNA (25 ng/condition) in the presence or absence of competitors. After washing, bound DNA was quantified by adding peroxidase-labeled anti-DIG antibodies and substrate. Results are expressed as mean absorbance +SD of triplicate values. Experiment is representative of two additional experiments. * P<0.001.

Article Snippet: The immunoprecipitation was performed using 4 μg of normal rabbit IgG (negative control), 10 μl of anti-PolII antibodies (positive control) and 4 μg of rabbit anti-TRF2 antibody (NB100-56694, Novus Biologicals) with an overnight incubation at 4°C.

Techniques: Recombinant, Incubation, Labeling, Binding Assay, Acrylamide Gel Assay

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) Schematic representation of the HHV-6A/B genome. The DR6 probe used for hybridization is shown in red. Uninfected and HHV-6A-infected HSB-2 cells (B-D) or uninfected and HHV-6B-infectd Molt-3 cells (E-F) were analyzed for TRF2 binding to viral DNA using ChIP. The input was hybridized with Alu probe to assess quantity of starting material. Anti-IgG (negative control), anti-PolII (positive control) or TRF2 antibodies were used for immunoprecipitation. B) QPCR detection of GAPDH DNA following ChIP. Results are expressed as fold increase over control IgG. C and E) Eluted DNA was hybridized with 32 P-labeled Alu, telomeric (TTAGGG) 3 or HHV-6A (DR6) probes. After hybridization the membranes were washed and exposed to X-ray films. D and F) Densitometric analysis of relative binding of TRF2 to telomeric and viral DNA. Results of one experiment representative of three are presented and are expressed as signal after normalization to input.

Article Snippet: The immunoprecipitation was performed using 4 μg of normal rabbit IgG (negative control), 10 μl of anti-PolII antibodies (positive control) and 4 μg of rabbit anti-TRF2 antibody (NB100-56694, Novus Biologicals) with an overnight incubation at 4°C.

Techniques: Hybridization, Infection, Binding Assay, Negative Control, Positive Control, Immunoprecipitation, Control, Labeling

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: U2OS cells were infected with HHV-6A and analyzed for TRF2 and IE2 expression at 24h, 48h and 72h post-infection by dual color immunofluorescence. A) Representative immunofluorescence of TRF2 and IE2 expression in bystander and IE2 expressing cells at 24, 48h and 72h post infection. B) Mean relative TRF2 expression ± SD in uninfected (blue), IE2- (green-uninfected bystander) or IE2+ (red-infected) cells at 24h, 48h and 72h post infection. Each symbol represents the relative TRF2 expression from a single nucleus.

Article Snippet: The immunoprecipitation was performed using 4 μg of normal rabbit IgG (negative control), 10 μl of anti-PolII antibodies (positive control) and 4 μg of rabbit anti-TRF2 antibody (NB100-56694, Novus Biologicals) with an overnight incubation at 4°C.

Techniques: Infection, Expressing, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) U2OS cells were mock-infected or infected with HHV-6A for 48h after which cells were processed for IF-FISH. Telomeres were labeled in magenta, TRF2 in green and IE2 in red. The panels in the middle row show images of cells with IE2 patches overlapping with large, diffuse TRF2 and telomeric staining (rectangles). The panels in the third row represent infected cells with punctate IE2 pattern colocalizing with TRF2 and telomeres (dashed squares). The colocalization of IE2, TRF2 and telomeres are shown in both 2D and 3D images. B) Uninfected and HHV-6A-infected U2OS cells were transfected with an empty vector, a myc-tagged-TRF1 expression vector. Forty-eight hours later cells were processed for IF-FISH. TRF1 was labeled in green and IE2 in red. Nuclei were stained with DAPI. Images on the far right show 2D colocalization of TRF1 with IE2.

Article Snippet: The immunoprecipitation was performed using 4 μg of normal rabbit IgG (negative control), 10 μl of anti-PolII antibodies (positive control) and 4 μg of rabbit anti-TRF2 antibody (NB100-56694, Novus Biologicals) with an overnight incubation at 4°C.

Techniques: Infection, Labeling, Staining, Transfection, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) A stick diagram of the IE2 protein with various domains identified is presented. B) Colocalization of HHV-6A IE2 protein with telomeres in the absence of viral DNA. U2OS cells were transfected with an empty vector, with IE2 expression vector or with IE2 Δ1290–1500 expression vector. Forty-eight hours later cells were processed for dual color immunofluorescence. Telomeres were labeled in cyan and IE2 in red. Nuclei are outlined by dashed lines. Examples of IE2 colocalizing with telomeres are presented in a 3D view (white arrows). C) The graph represents the mean ± SD % of WT IE2 and Δ1290–1500 IE2 localizing with telomeres. D) Lack of colocalization between HHV-6A p41 and telomeres in uninfected cells. U2OS cells were transfected with an empty vector or with a p41 expression vector. Forty-eight hours later cells were processed for dual color immunofluorescence. TRF2 was labeled green, p41 in red and nuclei outlined by a dashed line.

Article Snippet: The immunoprecipitation was performed using 4 μg of normal rabbit IgG (negative control), 10 μl of anti-PolII antibodies (positive control) and 4 μg of rabbit anti-TRF2 antibody (NB100-56694, Novus Biologicals) with an overnight incubation at 4°C.

Techniques: Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Labeling

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: U2OS cells were transduced with a lentiviral vector coding for a Dox inducible control shRNA (shCtrl) or a shRNA against TRF2 (shTRF2) and selected with puromycin +/- Dox for a week. A) Western blot analysis of TRF2 expression one week post selection. Membranes were also probed with anti-tubulin antibodies to show the input material loaded. B) One week post selection, +Dox cells were infected with HHV-6A for 48h and processed for IFA using anti-TRF2 (green) and anti-IE2 (red). Cells with IE2 in punctate form and cells with large patchy IE2, likely to represent VRC, are shown. Nuclei are outlined by dashed lines. C) The percentage of HHV-6A infected cells (from B) was estimated after counting a minimum of 700 cells and scoring the IE2 + ones. Results are expressed as mean %IE2 + cells ± SD. D) Mean percentage ± SD of IE2 localizing with telomeres in the presence (shCtrl +Dox) and absence (shTRF2 +Dox) of TRF2. Each dot represents the % of IE2 foci localizing with telomeres in one nucleus. ****p<0.0001. E) IF-FISH confocal images of shCtrl (+Dox) and shTRF2 (+Dox) cells analyzed for TRF2 (green), IE2 (red) and telomeres (cyan). Nuclei are outlined by dashed circles. Examples of IE2 localizing with telomeres (top row) or not found with telomeres (bottom row) are highlighted by the dashed polygons.

Article Snippet: The immunoprecipitation was performed using 4 μg of normal rabbit IgG (negative control), 10 μl of anti-PolII antibodies (positive control) and 4 μg of rabbit anti-TRF2 antibody (NB100-56694, Novus Biologicals) with an overnight incubation at 4°C.

Techniques: Transduction, Plasmid Preparation, Control, shRNA, Western Blot, Expressing, Selection, Infection

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) U2OS cells were transduced with lentiviral vectors expressing a scrambles shRNA (shCtrl) or a shTRF2. After a week of selection, cells were monitored for TRF2 expression by western blot. B) After a week of selection, shCtrl and shTRF2 treated cells were infected with HHV-6A or HHV-6B. After 30 days, DNA was isolated and the relative frequency of integration, relative to shCtrl set at 100%, estimated by ddPCR. *p<0.05.

Article Snippet: The immunoprecipitation was performed using 4 μg of normal rabbit IgG (negative control), 10 μl of anti-PolII antibodies (positive control) and 4 μg of rabbit anti-TRF2 antibody (NB100-56694, Novus Biologicals) with an overnight incubation at 4°C.

Techniques: Transduction, Expressing, shRNA, Selection, Western Blot, Infection, Isolation