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mouse monoclonal anti-trf2  (Becton Dickinson)

 
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    Becton Dickinson mouse monoclonal anti-trf2
    ( a ) Elevated levels of <t>TRF2</t> protein in a number of breast cancer and melanoma cells. Immunoblotting was performed to detect TRF2 in whole-cell extracts of the following human cell lines: Primary fibroblasts: IMR90, BJ and WI38; Breast cancer cells: MDA-MB-231, MDA-MB-453, MDA-MB-468, ZR-75-1, MCF-7 and SK-BR-3; Melanoma cells: Lox, CaCL 73-36, WM115, WM278, WM983A, WM983B and WM1158. Fibrosarcoma cell: HT1080. Tubulin was used as a loading control. ( b ) Assessing TRF2 overexpression levels. Parallel cultures of HT1080 clone A6 (a subclone of HT1080 cells that maintain stable telomere length) cells infected with lentiviruses expressing GFP or TRF2 were examined by immunoblotting (top panel) or immunostaining (bottom panel). Fold of TRF2 expression was quantified by the ImageJ software and normalized to tubulin levels. ( c ) Terminal Restriction Fragment analysis of HT1080 A6 cells infected with lentiviruses expressing GFP or TRF2. Cells were continuously passaged and collected at the indicated population doublings (PD). ( d ) Schematic diagram of STELA analysis. ( e ) Individual telomere lengths measured by STELA analysis in HT1080 A6 cells overexpressing GFP or TRF2 at PD6. Each lane represents a single PCR reaction performed with 100 pg of genomic DNA, followed by Southern blotting detection of XpYp telomeres using an XpYp subtelomeric probe.
    Mouse Monoclonal Anti Trf2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+monoclonal+anti+trf2/pmc04686832-131-5-8?v=Becton+Dickinson
    Average 90 stars, based on 1 article reviews
    mouse monoclonal anti-trf2 - by Bioz Stars, 2026-08
    90/100 stars

    Images

    1) Product Images from "Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions"

    Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

    Journal: Nature Communications

    doi: 10.1038/ncomms10132

    ( a ) Elevated levels of TRF2 protein in a number of breast cancer and melanoma cells. Immunoblotting was performed to detect TRF2 in whole-cell extracts of the following human cell lines: Primary fibroblasts: IMR90, BJ and WI38; Breast cancer cells: MDA-MB-231, MDA-MB-453, MDA-MB-468, ZR-75-1, MCF-7 and SK-BR-3; Melanoma cells: Lox, CaCL 73-36, WM115, WM278, WM983A, WM983B and WM1158. Fibrosarcoma cell: HT1080. Tubulin was used as a loading control. ( b ) Assessing TRF2 overexpression levels. Parallel cultures of HT1080 clone A6 (a subclone of HT1080 cells that maintain stable telomere length) cells infected with lentiviruses expressing GFP or TRF2 were examined by immunoblotting (top panel) or immunostaining (bottom panel). Fold of TRF2 expression was quantified by the ImageJ software and normalized to tubulin levels. ( c ) Terminal Restriction Fragment analysis of HT1080 A6 cells infected with lentiviruses expressing GFP or TRF2. Cells were continuously passaged and collected at the indicated population doublings (PD). ( d ) Schematic diagram of STELA analysis. ( e ) Individual telomere lengths measured by STELA analysis in HT1080 A6 cells overexpressing GFP or TRF2 at PD6. Each lane represents a single PCR reaction performed with 100 pg of genomic DNA, followed by Southern blotting detection of XpYp telomeres using an XpYp subtelomeric probe.
    Figure Legend Snippet: ( a ) Elevated levels of TRF2 protein in a number of breast cancer and melanoma cells. Immunoblotting was performed to detect TRF2 in whole-cell extracts of the following human cell lines: Primary fibroblasts: IMR90, BJ and WI38; Breast cancer cells: MDA-MB-231, MDA-MB-453, MDA-MB-468, ZR-75-1, MCF-7 and SK-BR-3; Melanoma cells: Lox, CaCL 73-36, WM115, WM278, WM983A, WM983B and WM1158. Fibrosarcoma cell: HT1080. Tubulin was used as a loading control. ( b ) Assessing TRF2 overexpression levels. Parallel cultures of HT1080 clone A6 (a subclone of HT1080 cells that maintain stable telomere length) cells infected with lentiviruses expressing GFP or TRF2 were examined by immunoblotting (top panel) or immunostaining (bottom panel). Fold of TRF2 expression was quantified by the ImageJ software and normalized to tubulin levels. ( c ) Terminal Restriction Fragment analysis of HT1080 A6 cells infected with lentiviruses expressing GFP or TRF2. Cells were continuously passaged and collected at the indicated population doublings (PD). ( d ) Schematic diagram of STELA analysis. ( e ) Individual telomere lengths measured by STELA analysis in HT1080 A6 cells overexpressing GFP or TRF2 at PD6. Each lane represents a single PCR reaction performed with 100 pg of genomic DNA, followed by Southern blotting detection of XpYp telomeres using an XpYp subtelomeric probe.

    Techniques Used: Western Blot, Over Expression, Infection, Expressing, Immunostaining, Software, Southern Blot

    ( a ) Representative metaphase spread image of HeLa1.2.11 cells infected with lentivirus expressing GFP or TRF2. Infected cells were passaged and collected at PD7 for metaphase spread followed by FISH analysis. Chromosomes (blue) were hybridized with PNA probes for telomeric sequences (green) or centromeric sequences (red). Regions in white boxes are enlarged to the bottom of the corresponding image for better visualization. Yellow arrows indicate signal-free telomeres; arrowhead indicates chromosome end-to-end fusions. For b and c , 50 metaphases (∼3,360 chromosomes) each of GFP- or TRF2-overexpressing cells were examined for telomeric abnormality. All quantifications were carried out blindly. Each point on the scatter plot represents a single metaphase. Mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( b ) Quantification of signal-free telomeres in HeLa1.2.11 cells overexpressing GFP or TRF2. ( c ) Quantification of chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing GFP or TRF2. ( d ) Schematic diagram of Fusion PCR analysis. ( e ) Individual chromosome end-to-end fusions assessed by Fusion PCR. HeLa1.2.11 cells overexpressing GFP or TRF2 were harvested at PD6. Multiple aliquots of 100 ng of genomic DNA were independently subjected to fusion PCR using a mix of XpYp, 17p and 21q subtelomeric primers. PCR products were resolved on 1% agarose-TBE gel and detected by Southern hybridization with an XpYp-specific subtelomeric probe. ( f ) Representative sequence of fusion molecules between XpYp, 17p and 21q. The fusion points, size of deletion, and microhomology (in red) are indicated.
    Figure Legend Snippet: ( a ) Representative metaphase spread image of HeLa1.2.11 cells infected with lentivirus expressing GFP or TRF2. Infected cells were passaged and collected at PD7 for metaphase spread followed by FISH analysis. Chromosomes (blue) were hybridized with PNA probes for telomeric sequences (green) or centromeric sequences (red). Regions in white boxes are enlarged to the bottom of the corresponding image for better visualization. Yellow arrows indicate signal-free telomeres; arrowhead indicates chromosome end-to-end fusions. For b and c , 50 metaphases (∼3,360 chromosomes) each of GFP- or TRF2-overexpressing cells were examined for telomeric abnormality. All quantifications were carried out blindly. Each point on the scatter plot represents a single metaphase. Mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( b ) Quantification of signal-free telomeres in HeLa1.2.11 cells overexpressing GFP or TRF2. ( c ) Quantification of chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing GFP or TRF2. ( d ) Schematic diagram of Fusion PCR analysis. ( e ) Individual chromosome end-to-end fusions assessed by Fusion PCR. HeLa1.2.11 cells overexpressing GFP or TRF2 were harvested at PD6. Multiple aliquots of 100 ng of genomic DNA were independently subjected to fusion PCR using a mix of XpYp, 17p and 21q subtelomeric primers. PCR products were resolved on 1% agarose-TBE gel and detected by Southern hybridization with an XpYp-specific subtelomeric probe. ( f ) Representative sequence of fusion molecules between XpYp, 17p and 21q. The fusion points, size of deletion, and microhomology (in red) are indicated.

    Techniques Used: Infection, Expressing, Two Tailed Test, Hybridization, Sequencing

    ( a ) Formation of thinly stretched telomere bridges between anaphase chromosomes in HeLa1.2.11 cells overexpressing TRF2. Telomeric DNAs were detected by in situ hybridization with a PNA telomeric probe (red). Chromosomes were stained with DAPI (blue). ( b ) Quantification of telomeric anaphase bridges and chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing TRF2 at PD3 and PD7. HeLa1.2.11 cells were infected with lentiviruses expressing TRF2. Parallel cultures were collected at PD3 or PD7 for PNA telomere-FISH to examine telomeric anaphase bridges or for metaphase spreading followed by PNA telomere-FISH to examine chromosome end-to-end fusions. ( c ) Quantification of fragile telomeres in HeLa1.2.11 cells overexpressing GFP control or TRF2 at PD3. Cells were collected for metaphase spreading followed by PNA telomere-FISH to examine fragile telomeres. Representative fragile telomeres are labelled by yellow arrows on images at the left panel. All quantifications were carried out blindly. For b and c , mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( d ) TRF2 overexpression stalled replication at telomeres. Representative chromatin fibre FISH images showed the incorporation of IdU (blue) or CldU (green) at telomeric (red) and adjacent subtelomeric regions in LOX cells infected with lentiviruses expressing luciferase control or TRF2. At PD3, cells in logarithmic growth were labelled sequentially with 30 μM of IdU and then CldU for 4 h each before Chromatin fibre-FISH analysis was carried out. Telomeres were identified by FISH with a telomeric repeat probe. IdU and CldU were identified by immunostaining with analogue-specific antibodies. Dotted line represents the start of telomeric sequences. We did not see dual IdU and CldU labelling at replicating telomeres due to the long labelling time (4 h) used for each halogenated nucleotide. ( e ) Quantification of fraction of telomeric fragments that was labelled with CldU and/or IdU. For control of stalled replication, cells were treated with 1 μg ml −1 aphidicolin for 16 h before they were labelled with IdU and CldU in the presence of aphidicolin (see for representative images).
    Figure Legend Snippet: ( a ) Formation of thinly stretched telomere bridges between anaphase chromosomes in HeLa1.2.11 cells overexpressing TRF2. Telomeric DNAs were detected by in situ hybridization with a PNA telomeric probe (red). Chromosomes were stained with DAPI (blue). ( b ) Quantification of telomeric anaphase bridges and chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing TRF2 at PD3 and PD7. HeLa1.2.11 cells were infected with lentiviruses expressing TRF2. Parallel cultures were collected at PD3 or PD7 for PNA telomere-FISH to examine telomeric anaphase bridges or for metaphase spreading followed by PNA telomere-FISH to examine chromosome end-to-end fusions. ( c ) Quantification of fragile telomeres in HeLa1.2.11 cells overexpressing GFP control or TRF2 at PD3. Cells were collected for metaphase spreading followed by PNA telomere-FISH to examine fragile telomeres. Representative fragile telomeres are labelled by yellow arrows on images at the left panel. All quantifications were carried out blindly. For b and c , mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( d ) TRF2 overexpression stalled replication at telomeres. Representative chromatin fibre FISH images showed the incorporation of IdU (blue) or CldU (green) at telomeric (red) and adjacent subtelomeric regions in LOX cells infected with lentiviruses expressing luciferase control or TRF2. At PD3, cells in logarithmic growth were labelled sequentially with 30 μM of IdU and then CldU for 4 h each before Chromatin fibre-FISH analysis was carried out. Telomeres were identified by FISH with a telomeric repeat probe. IdU and CldU were identified by immunostaining with analogue-specific antibodies. Dotted line represents the start of telomeric sequences. We did not see dual IdU and CldU labelling at replicating telomeres due to the long labelling time (4 h) used for each halogenated nucleotide. ( e ) Quantification of fraction of telomeric fragments that was labelled with CldU and/or IdU. For control of stalled replication, cells were treated with 1 μg ml −1 aphidicolin for 16 h before they were labelled with IdU and CldU in the presence of aphidicolin (see for representative images).

    Techniques Used: In Situ Hybridization, Staining, Infection, Expressing, Two Tailed Test, Over Expression, Luciferase, Immunostaining

    ( a ) Representative anaphase images showing the staining of PICH, telomeres, and centromeres in HeLa1.2.11 cells overexpressing TRF2. Cells were infected with lentivirus expressing TRF2 and collected at PD2 after infection for immunostaining-FISH analysis. Telomeres (magenta) and centromeres (red) were identified by PNA FISH. PICH (green) were identified by immunostaining with an anti-PICH antibody. Chromosomes were stained with DAPI (blue). PICH-aligned telomeric anaphase bridges were marked by white arrows. Note that the image represents a single section on the z axis. ( b ) Quantification of PICH bridges, as well as centromere-associated PICH bridges, in HeLa1.2.11 cells overexpressing GFP control or TRF2. Bars represent mean values and s.e.m. (>150 anaphases from three independent experiments examined for each line). Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( c ) Quantification of fraction of telomeric UFBs that associate with the PICH protein. 75 telomeric UFB-containing anaphases from three independent experiments were examined for the association between PICH and telomeric UFB. ( d ) Representative anaphase images showing the staining of PICH and centromeres in HeLa1.2.11 cells treated with 20 μM DNA topoisomerase II inhibitor ICRF-159.
    Figure Legend Snippet: ( a ) Representative anaphase images showing the staining of PICH, telomeres, and centromeres in HeLa1.2.11 cells overexpressing TRF2. Cells were infected with lentivirus expressing TRF2 and collected at PD2 after infection for immunostaining-FISH analysis. Telomeres (magenta) and centromeres (red) were identified by PNA FISH. PICH (green) were identified by immunostaining with an anti-PICH antibody. Chromosomes were stained with DAPI (blue). PICH-aligned telomeric anaphase bridges were marked by white arrows. Note that the image represents a single section on the z axis. ( b ) Quantification of PICH bridges, as well as centromere-associated PICH bridges, in HeLa1.2.11 cells overexpressing GFP control or TRF2. Bars represent mean values and s.e.m. (>150 anaphases from three independent experiments examined for each line). Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( c ) Quantification of fraction of telomeric UFBs that associate with the PICH protein. 75 telomeric UFB-containing anaphases from three independent experiments were examined for the association between PICH and telomeric UFB. ( d ) Representative anaphase images showing the staining of PICH and centromeres in HeLa1.2.11 cells treated with 20 μM DNA topoisomerase II inhibitor ICRF-159.

    Techniques Used: Staining, Infection, Expressing, Immunostaining, Two Tailed Test

    ( a ) Long telomere length exacerbates TRF2-induced telomeric UFBs. Left panel: quantification of telomeric UFBs in cells with different mean telomere lengths. Approximately 50 anaphases from each cell line were analysed for telomeric UFBs. Mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. No telomeric UFBs were detected in HeLa1.2.11 and HT1080 cells infected with lentivirus overexpressing GFP. Telomeres in UM-UC-3 cells (∼3 kb) were pre-extended by expression of the telomerase RNA subunit (hTR) for 11 days (to ∼8 kb) and 20 days (to ∼15 kb). Cells were infected with lentivirus expressing TRF2, and then fixed for telomeric FISH 48 h after infection. TRF2 overexpression did not induce any telomeric UFBs in control UM-UC-3 cells that were infected with an empty lentiviral vector. Right panel: terminal restriction fragment analysis in UM-UC-3 cells expressing vector control or hTR using a telomeric repeat probe. ( b ) TRF2 overexpression fails to induce telomere shortening in cells containing very short telomere length.
    Figure Legend Snippet: ( a ) Long telomere length exacerbates TRF2-induced telomeric UFBs. Left panel: quantification of telomeric UFBs in cells with different mean telomere lengths. Approximately 50 anaphases from each cell line were analysed for telomeric UFBs. Mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. No telomeric UFBs were detected in HeLa1.2.11 and HT1080 cells infected with lentivirus overexpressing GFP. Telomeres in UM-UC-3 cells (∼3 kb) were pre-extended by expression of the telomerase RNA subunit (hTR) for 11 days (to ∼8 kb) and 20 days (to ∼15 kb). Cells were infected with lentivirus expressing TRF2, and then fixed for telomeric FISH 48 h after infection. TRF2 overexpression did not induce any telomeric UFBs in control UM-UC-3 cells that were infected with an empty lentiviral vector. Right panel: terminal restriction fragment analysis in UM-UC-3 cells expressing vector control or hTR using a telomeric repeat probe. ( b ) TRF2 overexpression fails to induce telomere shortening in cells containing very short telomere length.

    Techniques Used: Two Tailed Test, Infection, Expressing, Over Expression, Plasmid Preparation



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    Figure Lengend Snippet: a The diagram shows the workflow on a timeline: on the day after cell seeding, cells were treated with RHPS4 (0.2 and/or 0.5 μM). At 48 h after treatment, cells were again treated with RHPS4 (0.2 and/or 0.5 μM) or harvested and counted. At 72 h after the first treatment, the cell viability was checked, and finally at 96 h after the first treatment, the cells were harvested and processed. b Representative images showing U251MG cells stained by immunofluorescence using anti-BLM (red signals), and anti-TRF1 or anti-TRF2 (green signals) antibodies. Merged images allow visualization of colocalizing dots (yellow signals). Yellow arrows indicate BLM and TRF1-TRF2 colocalizations. c Quantification of the colocalizations between BLM and TRF1 or TRF2 proteins in U251MG cell line upon RHPS4 treatment (0.2 and 0.5 μM). d , e Sensitivity of U251MG and BLM −/− cell lines to RHPS4 concentrations ranging from 0.01 to 2 μM (0.01; 0.125; 0.25; 0.5; 1; 2 μM), evaluated 96 h after the first treatment. Mitomycin C (MMC) was used as a positive control at concentrations of 0.1; 0.5; 1; 2; 5 μg/ml. The Sulforhodamine B (SRB) cytotoxicity assay showed that RHPS4 sensitivity was unchanged in U251MG and BLM −/− (IC50 was 0.56 μM in both cell lines). f Short-term cell proliferation in untreated cells, U251MG and BLM −/− , and in RHPS4-treated cells (0.5 μM) as evaluated 48 and 96 h after the first treatment. g Long-term cell proliferation in U251MG and BLM −/− untreated and RHPS4-treated cells (0.5 μM). Scale bars represent 5 μm. * p < 0.05, ** p < 0.01 (two-way ANOVA; n = 3). Error bars denote the standard deviation of the mean.

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