mouse anti trf2 img 124a  (Novus Biologicals)

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: (A) Summary of the doubly genome-edited cell line generated to track telomeres and single telomerase RNPs and to visualize Cajal bodies. TRF2 and TERT were fused N-terminally with HA-mEos3.2 tag and FLAG-HaloTag, respectively. BFP-coilin was transiently expressed. (B) Western blot and fluorescence imaging of TERT immuno-purified from parental HeLa and three genome-edited cells lines, using FLAG and TERT antibodies (* tagged TERT, # endogenous TERT). The HaloTag and SNAP-tag were labeled with JF646. (C) Direct telomerase extension assay after immuno-purification of TERT. LC1 and LC2, labeled DNA loading controls. (D) Western blot of TERT purifications used for the telomerase assay shown in panel C (* tagged TERT, # endogenous TERT). (E) Cyto-localization of FLAG-HaloTag-TERT in fixed cells synchronized in S-phase. TERT was labeled with JF646; Cajal bodies and telomeres were stained with antibodies against coilin and TRF2, respectively (white arrows indicate co-localizations). See also Figures S1 and S2.

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Generated, Western Blot, Fluorescence, Imaging, Purification, Labeling, Telomerase Assay, Staining

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: (A) Cyto-localization of K78E FLAG-HaloTag-TERT in fixed cells synchronized in S-phase. TERT was labeled with JF646; Cajal bodies and telomeres were stained with antibodies against coilin and TRF2, respectively. K78E TERT associates with Cajal bodies but not telomeres. (B–D) Comparison of the survival probabilities of stationary wild-type and K78E telomerase particles at different nuclear locations: (B) K78E telomerase particles at telomeres, Cajal bodies, and other nuclear sites, (C) Wild-type and K78E TERT at telomeres, (D) Wild-type and K78E TERT at Cajal bodies and other nuclear sites (N = 18 cells for wild-type, N = 14 cells for K78E TERT). K78E TERT particles at telomeres have dynamics indistinguishable from those at other nuclear locations, and they have a reduced survival probability compared to wild-type TERT. In contrast, survival probabilities at Cajal bodies and other nuclear locations are identical for both TERT proteins (also see Fig. S3F–H). (E) Diffusion coefficient histogram of telomeric K78E telomerase tracks present for at least 5 consecutive frames (N=14 cells). K78E telomerase lacks the highly static population (D ~ 0.001 μm2/s). See also Figure S3 and Movie S8.

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Labeling, Staining, Comparison, Diffusion-based Assay

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: (A) Diagram illustrating our approach for visualizing Cajal bodies, telomerase, and telomeres. First, BFP-coilin was imaged, also converting mEOS-3.2 from the green to the red state. Immediately afterwards, we simultaneously imaged red mEOS3.2-TRF2 and HaloTag(JF646)-TERT at 45 frames per second. (B) Still images from movies simultaneously visualizing telomeres (mEOS3.2) and telomerase (HaloTag-JF646), after imaging Cajal bodies marked by BFP-coilin (white arrows indicate co-localizations). (C) A subset of trajectories of telomerase particles, present for at least 10 frames (~220 ms), generated by single-particle tracking of telomerase signals at 45 fps (20 ms exposure), demonstrating rapid three-dimensional diffusion. (D) All TERT trajectories detected in a 45 s movie. Unexplored region marked by asterisk. (E) Diffusion coefficient histogram of telomerase tracks present for at least 5 consecutive frames (N = 18 cells, n = 5035 tracks). Two freely diffusing populations (D ~ 0.3 μm2/s, magenta; D ~ 1.3 μm2/s, blue) and a smaller less mobile population (D ~ 0.01 μm2/s, green) are present. Fractions of the total number of particles in each population are indicated. Also see Movies S1–S4.

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Imaging, Generated, Single-particle Tracking, Diffusion-based Assay

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: Kymographs of telomerase tracks overlapping with TRF2 signals, showing (A) static long-lasting and (B) dynamic “probing” interactions. (C) Approach to analyze static TERT particles. Each TERT track segment represents the distance traveled in a single time interval (~22 ms). Large distances correspond to higher diffusion coefficients. When a binding event occurs, TERT signals remain largely static for several consecutive frames (arrows). By constraining the diffusion coefficient of the tracking algorithm to D < 0.05 μm2/s, only stationary TERT trajectories are analyzed. The length of these tracks corresponds to the time the particle is bound to a particular nuclear locus. (D) Quantification of the survival probability of stationary telomerase particles associated with telomeres, Cajal bodies and nuclear sites (N = 18 cells). TERT particles associate with Cajal bodies and telomeres for longer times than with other nuclear loci. (E) Frequency distribution of the occurrence of stationary particles per telomere in a 45 s timespan (N = 18 cells). (F) Distribution of the duration of long, static interactions between telomerase and telomeres (N = 13 cells, n = 29 interactions, horizontal bar = median, box = 25–75% percentile, whiskers = min-max). Also see Movies S5–S7.

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Diffusion-based Assay, Binding Assay

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: (A) Approach to spatially segregate telomerase tracks. Tracks were assigned to telomeres or Cajal bodies if they came within 3 pixels (0.48 μm) of the centroid of a TRF2 signal or 5 pixels (0.8 μm) of the centroid of a coilin signal, respectively. (B) Diffusion coefficient histograms of telomerase trajectories close to telomeres, Cajal bodies, or neither, present for at least 5 consecutive frames (N = 18 cells). Cajal body associated tracks are enriched for a less mobile population (D ~ 0.01 μm2/s), while telomere associated tracks instead display a highly static population (D ~ 0.001 μm2/s). Fractions of the total number of particles in each population are indicated. (C) Diffusion coefficient histogram of TRF2 tracks. Telomeres move with the same diffusion coefficient as the highly static telomere-associated TERT population (D ~ 0.001 μm2/s).

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Diffusion-based Assay

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Recombinant, Sequencing, Software

Journal: Genome Biology

Article Title: A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression

doi: 10.1186/s13059-015-0791-1

Figure Lengend Snippet: TERT localizes to telomeres in S but not G1 phase of the cell cycle. a IF analysis of fixed HeLa cells expressing FLAG-SNAP-TERT, synchronized in G1 and S phase of the cell cycle (scale bar = 5 μm). Cells expressing FLAG-SNAP-TERT displayed telomere-localized TERT foci in S but not G1 phase of the cell cycle, while parental cells never showed telomere-localized TERT foci. b FACS analysis of the DNA content of cells synchronized in S phase showed a peak between the 2 N and 4 N peaks of asynchronous cells, confirming that they were in S phase. The G1 cell population contained 2 N and 4 N peaks but was depleted for cells with intermediate DNA content. 4 N cells, which failed to release from their mitotic arrest, were easily distinguished from G1 cells by their morphology. c Quantification of the number of TERT foci which co-localized with TRF2 signals in edited HeLa cells synchronized at different stages of the cell cycle. Data were generated from two independent experiments, each analyzing 50 cells per condition (mean ± standard deviation). d FACS analysis of the DNA content of edited HeLa cells released from a double thymidine block as they transition through S phase. Prior to release, the cell population contained mostly cells with 2 N DNA content, which progressively increased as the cells underwent DNA replication. Nine to ten hours after release, DNA replication was complete, as indicated by the majority of cells having 4 N DNA content. e Quantification of the number of TERT foci co-localized with TRF2 signals at different time points during S phase (50 cells per time point, mean ± standard error of the mean; for corresponding images see Fig. S4 in Additional file ). A.U. arbitrary units, Propidium Iodide (PI)

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature, and incubated in blocking buffer (3 % BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) and coilin (Santa Cruz, sc-32860, 1:100) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies, A-31556, and Abcam, ab150117, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong® Diamond Antifade Mountant (Life Technologies, P36970).

Techniques: Expressing, Generated, Standard Deviation, Blocking Assay

Journal: Journal of Cell Science

Article Title: Role of progerin-induced telomere dysfunction in HGPS premature cellular senescence

doi: 10.1242/jcs.067306

Figure Lengend Snippet: Effect of progerin on the association of telomere-binding proteins with telomeric DNA. (A) NDFs (IMR90) were infected with viruses expressing progerin, TRF2ΔBΔM, or vector control and marker selected for 7 days. For ChiP analysis, antibodies specific for TRF1, TRF2 or an IgG negative control were used for immunoprecipitation following crosslinking and DNA shearing. DNA was loaded onto a nylon membrane using a slot blot and hybridized with a DIG-labeled telomere probe. Input DNA represents 1% of total DNA. (B) Quantification of blot shown in A. Signal density was measured by ImageJ, and histogram values represent the TRF1 and TRF2 telomeric ChIP signal normalized to input signal. The amount of telomeric DNA in each ChIP is expressed in arbitrary units (a.u.) after vector control sequences were normalized to 1. (C) NDFs (IMR90) were infected with lentiviruses expressing progerin or vector control and marker selected for 5 days. For ChiP analysis, antibodies specific for TRF1, TRF2 or an IgG negative control were used for immunoprecipitation following crosslinking and DNA shearing. DNA was loaded onto a nylon membrane using a slot blot and hybridized with a DIG-labeled telomere probe, stripped and re-hybridized with a DIG-labeled Alu probe. Input DNA represents 1% of total DNA. (D) Quantification of blot in C performed as described in B.

Article Snippet: Antibodies against the following proteins were used: Lamin A/C (Millipore, MAB3211), p53 (1801, Mount Sinai School of Medicine Hybridoma Center), p21 (BD Biosciences, 556431), p16 (Santa Cruz, sc-468), Rb (Cell Signaling, 9309), Ser139 γH2AX (Millipore, 05-636), Ser139 γH2AX (Millipore, 07-164), Ser-1981 phospho-ATM (Rockland, 600-401-400), TERT (abcam, ab32020), TRF1 (#370) (a kind gift from Titia de Lange, Rockefeller University, NY), TRF1 (Santa Cruz, sc-6165), TRF2 (Imgenex, IMG-124A), β-actin (Sigma, A5441) and IgG (Millipore, 12-371).

Techniques: Binding Assay, Infection, Expressing, Plasmid Preparation, Control, Marker, Negative Control, Immunoprecipitation, Membrane, Dot Blot, Labeling

Journal:

Article Title: Telomerase-associated Protein 1, HSP90, and Topoisomerase II? Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates * S⃞

doi: 10.1074/jbc.M900195200

Figure Lengend Snippet: Identification of BLM- and TRF2-associated proteins by mass spectrometry. BLM-associated proteins were immunoprecipitated from immortalized telomerase-positive (HeLa and MCF7, lanes 3 and 5) and telomerase-negative ALT cells (WI38-VA13 and Saos2, lanes 4 and 6) using anti-BLM antibody. Lysates were subjected to a second round of immunoprecipitation using anti-TRF2 to enrich for BLM-associated proteins in cells that use ALT. Lanes 1 and 2 are IgG controls for MCF7 and Saos2 cells. Immunoprecipitates were resolved by 10% SDS-PAGE and silver-stained. Proteins identified by mass spectrometry are labeled.

Article Snippet: Antibodies were purchased from Accurate Scientific (BrdUrd, OBT 0030), Bethyl Laboratories (BLM, A300-110A for immunoprecipitations and Western blots; TOPOIIα, A300-054A for immunoprecipitations and Western blots), Calbiochem (TOPOIIα, NA14 for immunofluorescence), Imgenex (TRF2, IMG-124A for immunofluorescence), and Santa Cruz Biotechnology (HSP90, sc-1057; PML, PG-M3; TEP1, sc-13052).

Techniques: Mass Spectrometry, Immunoprecipitation, SDS Page, Staining, Labeling

Journal:

Article Title: Telomerase-associated Protein 1, HSP90, and Topoisomerase II? Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates * S⃞

doi: 10.1074/jbc.M900195200

Figure Lengend Snippet: Detection of BLM and TRF2-associated proteins by Western analysis. A, BLM and TRF2 were coimmunoprecipitated sequentially from nuclear extracts of ALT (WI38-VA13 and Saos2) and telomerase-positive (HeLa and MCF7) or BS cells (GM08508) (panels a–c). Immunoprecipitations (IP) were carried out with goat polyclonal BLM and TRF2 antibodies or normal goat IgG (negative control). Immunoprecipitates were resolved by 6% SDS-PAGE, Western-blotted with anti-TEP1 (a), anti-TOPOIIα (b), and anti-HSP90 (c) antibodies. TEP1 (d), TOPOIIα (e), and HSP90 (f) were also immunoprecipitated from nuclear extracts of Saos2 and MCF7 cells, resolved, and probed with anti-BLM and anti-TRF2. IgG controls and the inputs are also shown. B, TRF1 associates with BLM and TRF2. BLM·TRF2 immunocomplex from Saos2 and MCF7 were subjected to Western analysis using anti-TRF1 antibody. TRF1 immunoprecipitated from the ALT cell line Saos2. BLM was detected in the immunocomplex from both cell lines, whereas TRF2 was present in complex with BLM and TRF1 in Saos2. C, TEP1 is not a component of the ALT complex containing TRF1. Whole cell lysates from Saos2 and MCF7 were subjected to sequential immunoprecipitation using anti-BLM and anti-TRF1 antibodies. HSP90, TOPOIIα, and TRF2 were detected in complex with BLM and TRF2 in Saos2 but not in MCF7. TEP1 was not included in this specific complex in either cell line. The Input lanes show the presence of the proteins in whole cell lysates.

Article Snippet: Antibodies were purchased from Accurate Scientific (BrdUrd, OBT 0030), Bethyl Laboratories (BLM, A300-110A for immunoprecipitations and Western blots; TOPOIIα, A300-054A for immunoprecipitations and Western blots), Calbiochem (TOPOIIα, NA14 for immunofluorescence), Imgenex (TRF2, IMG-124A for immunofluorescence), and Santa Cruz Biotechnology (HSP90, sc-1057; PML, PG-M3; TEP1, sc-13052).

Techniques: Western Blot, Negative Control, SDS Page, Immunoprecipitation

Journal:

Article Title: Telomerase-associated Protein 1, HSP90, and Topoisomerase II? Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates * S⃞

doi: 10.1074/jbc.M900195200

Figure Lengend Snippet: TEP1, TOPOIIα, and HSP90 co-localize with BLM and TRF2 in ALT cells in vivo. Three proteins co-localize with BLM (A) and TRF2 (B) in immortalized ALT cells Saos2. A, for generating immunofluorescence with BLM, cells were transfected with pEGFP-BLM, fixed, and immunostained using rabbit polyclonal anti-TEP1, anti-TOPOIIα, and anti-HSP90, and rhodamine-labeled secondary. Negative controls with rabbit IgG are shown. DAPI, 4′,6-diamidino-2-phenylindole. B, immunofluorescence with TRF2- and BLM-associated ATL complex proteins were performed as described under “Experimental Procedures.” Immunostaining was performed using mouse monoclonal anti-TRF2 and rabbit polyclonal antibodies against the BLM-associated proteins. Mouse IgG was used for negative controls. BLM (A) and TRF2 (B) merge with TEP1 or TOPOIIα or HSP90 to give yellow foci in ALT cell line Saos2. Strong co-localization was not observed in the telomerase-positive cell line MCF7. C, quantitation of A and B. % of cells showing co-localization of BLM or TRF2 with the BLM-associated ALT complex proteins were calculated from three independent experiments to obtain averages and S.D.

Article Snippet: Antibodies were purchased from Accurate Scientific (BrdUrd, OBT 0030), Bethyl Laboratories (BLM, A300-110A for immunoprecipitations and Western blots; TOPOIIα, A300-054A for immunoprecipitations and Western blots), Calbiochem (TOPOIIα, NA14 for immunofluorescence), Imgenex (TRF2, IMG-124A for immunofluorescence), and Santa Cruz Biotechnology (HSP90, sc-1057; PML, PG-M3; TEP1, sc-13052).

Techniques: In Vivo, Immunofluorescence, Transfection, Labeling, Immunostaining, Quantitation Assay

Journal:

Article Title: Telomerase-associated Protein 1, HSP90, and Topoisomerase II? Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates * S⃞

doi: 10.1074/jbc.M900195200

Figure Lengend Snippet: BLM knockdown and TRF length analysis in cells using ALT (Saos2) or telomerase (MCF7) to maintain telomeres demonstrate the requirement for BLM in ALT. A, Saos2 and MCF7 cells were transfected with pBLMsiRNA and control pSCsiRNA (negative or untreated control) and cultured for 3 weeks in the presence of the selective antibiotic puromycin (6 ng/μl). Stably transfected clones were harvested and analyzed by Western analysis using rabbit anti-BLM (Bethyl Laboratories). B, genomic DNAs were isolated and analyzed by Southern blot. TRF lengths are decreased in average size in Saos2 cells after siRNA treatment against BLM, in comparison to the treated controls. This decrease was not observed in MCF7 cells treated similarly. Size markers are shown to the left. Kbp, kilobase pairs. C, BLM knockdown affects the co-localization of the BLM-associated proteins with APBs and TRF2 in ALT cells. Co-localization of TEP1, TOPOIIα, and HSP90 with APBs or TRF2 was monitored by immunofluorescence in the ALT cell line Saos2 transiently transfected with control or pBLMsiRNA as described under “Experimental Procedures.” Percent co-localization was determined as before (Fig. 3C). D, BLM knockdown does not affect the expression level of TEP1, HSP90, and TOPOIIα. Total cell lysates prepared from control or pBLMsiRNA-transfected Saos2 and MCF7 cell lines were resolved by 10% SDS-PAGE and Western-analyzed using anti-TEP1, anti-HSP90, and anti-TOPOIIα. α-Actin was used for the loading control.

Article Snippet: Antibodies were purchased from Accurate Scientific (BrdUrd, OBT 0030), Bethyl Laboratories (BLM, A300-110A for immunoprecipitations and Western blots; TOPOIIα, A300-054A for immunoprecipitations and Western blots), Calbiochem (TOPOIIα, NA14 for immunofluorescence), Imgenex (TRF2, IMG-124A for immunofluorescence), and Santa Cruz Biotechnology (HSP90, sc-1057; PML, PG-M3; TEP1, sc-13052).

Techniques: Transfection, Cell Culture, Stable Transfection, Clone Assay, Western Blot, Isolation, Southern Blot, Comparison, Immunofluorescence, Expressing, SDS Page

Journal: Mobile DNA

Article Title: Creation of a novel telomere-cutting endonuclease based on the EN domain of telomere-specific non-long terminal repeat retrotransposon, TRAS1

doi: 10.1186/1759-8753-1-13

Figure Lengend Snippet: Subcellular localization of expressed chimeric endonucleases . (a) Telomere localization of expressed chimeric endonucleases. We infected adenovirus that expresses a chimeric endonuclease at MOI 100 to U2OS cells and performed indirect immunofluorescence the next day. Fusion proteins were detected with anti-HA antibody (green), and telomere was detected with anti-TRF2 antibody (red). Nuclei were stained with Hoechst33342. Yellow dot signals represent the colocalization of fusion protein and telomere. The scale bar is 10 μm. (b) Telomere cleavage by expressed chimeric endonucleases. In order to visualize telomere cleavage by chimeric endonucleases, fusion proteins were detected with anti-HA antibody (green) and DNA double-strand breaks were detected with anti-γ-H2AX antibody (red). (c) Based on the results in (b), we calculated the ratio (%) of co-localized spots (yellow) to fusion protein signals (green) using ImageJ (bar: 95% confidence interval). * shows a statistically significant increase of colocalization to ENmut-T ( P < 0.01).

Article Snippet: Anti-TRF2 antibody (IMGENEX, CA, USA; #IMG-124A) was used to detect telomere, because TRF2 is another telomere binding protein being independent of TRF1, and anti-HA antibody (BETHYL, TX, USA; #A190-108A) was used to detect chimeric endonucleases.

Techniques: Infection, Immunofluorescence, Staining