mammalian expression vector peg bacmam  (Addgene inc)

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 TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the Maltose-Binding Protein (MBP) coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

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 TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the Maltose-Binding Protein (MBP) coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Hybridization, Infection, Binding Assay, Negative Control, Positive Control, Immunoprecipitation, 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 TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the Maltose-Binding Protein (MBP) coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

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 TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the Maltose-Binding Protein (MBP) coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

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 TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the Maltose-Binding Protein (MBP) coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

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 TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the Maltose-Binding Protein (MBP) coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

Techniques: Transduction, Plasmid Preparation, 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 TRF2 coding sequence was excised from pLPC-NMYC TRF2 vector using with BamHI and XhoI and cloned in frame with the Maltose-Binding Protein (MBP) coding sequence of the pMAL-C2 vector (New England Biolabs) using BamHI and SalI enzymes.

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