Journal: Nature Communications

Article Title: TRF1 relies on fork reversal to prevent fragility at human telomeres

doi: 10.1038/s41467-025-61828-5

Figure Lengend Snippet: A Above: Representative blot from ChIP analyses showing TRF1 binding at telomeres in PCS ST and PCS LT cells and in PCS LT cells transduced with pLPC empty vector (EV) or pLPC-TRF1 (TRF1). Below: Quantifications. Signals were normalized first to telomeric input signals and then to either PCS LT (left part) or EV-transduced PCS LT cells (right part). n = 3 independent biological replicates for PCS LT and PCS ST and n = 4 independent biological replicates for PCS LT cells transduced with either EV or TRF1. Mean ± SEM. Two-tailed ratio paired t -tests. B Western blot of cell extracts from PCS LT cells transduced with pLPC/pLHCX-empty vector (EV), pLPC-TRF1 (TRF1) or pLHCX-RNaseH1-Myc (RH1), probed for TRF1 and Myc. Representative of n = 3 biological replicates. C Above: Representative CO-FISH pictures on metaphase spreads from PCS LT cells transduced with either EV, TRF1, or both TRF1 and RH1 showing lagging (green) and leading (red) telomere fragility (arrowheads). Below: Lagging (green) and leading (black) telomere fragility. A minimum of 5200 chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Kruskal–Wallis tests. Source data are provided as a Source Data file.

Article Snippet: One hundred microliters of lysate were kept as input and 900 μl were incubated with 1 μg of anti-TRF1 antibody (St John’s laboratory) for 4 h at 4 °C.

Techniques: Binding Assay, Transduction, Plasmid Preparation, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: TRF1 relies on fork reversal to prevent fragility at human telomeres

doi: 10.1038/s41467-025-61828-5

Figure Lengend Snippet: A Above: Western blot of cell extracts from PCS LT cells transduced with pLPC empty vector (EV) or pLPC-TRF1 (TRF1), probed for pRPA32-S33, RPA32, and Actin. Below: pRPA32-S33 (purple) and RPA32 (green) levels, normalized to Actin and to PCS LT -EV. Representative of n = 6 independent biological replicates. Mean ± SEM. Two-tailed ratio paired t -tests. B Co-localization events between telomeres and RAD51 (cyan) or RPA32 (green) in PCS LT cells transduced with either EV or TRF1. n nuclei were analyzed from three independent biological replicates. Median in red, quartiles in orange. Two-tailed Mann–Whitney tests. C Representative blot and quantification for CCA in PCS LT cells transduced with either EV or TRF1. Signals were normalized to PCS LT -EV. n = 3 independent biological replicates. Mean ± SEM. Two-tailed ratio paired t -test. D Co-localization events between telomeres and PML in PCS LT cells transduced with either EV or TRF1. n nuclei were analyzed from three independent biological replicates. Median in red, quartiles in orange. Two-tailed Mann–Whitney test. E Above left: Schematic representation of the pTelN mini-chromosome containing 115 telomeric repeats (red arrow) and the SV40 replication origin (ORI). Above right: Schematic representation of the 2D-gel migration profile of the specified DNA replication intermediates. Below left: 2D-gel analysis of plasmid DNA using the 5.2 kb Bam HI- Sac I probe, in cells with and without ectopic overexpression of TRF1. Below right: Quantification of cone signal (reversed fork migration) as a ratio of cone signal to total replication intermediates (RI), normalized to EV. n = 3 independent biological replicates. Mean ± SEM. Two-tailed ratio paired t -test. F , G Lagging (green) and leading (black) telomere fragility assessed by CO-FISH in PCS LT -EV or PCS LT -TRF1 cells under the specified conditions. siSM1: siSMARCAL1. Cells were treated with either 15 µM BIBR ( F ) or 5 µM Olaparib (PARPi) ( G ), or DMSO as control, during 48 h before BrdU/BrdC incorporation. A minimum of 8600 ( F ) or 6200 ( G ) chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Kruskal–Wallis tests. Source data are provided as a Source Data file.

Article Snippet: One hundred microliters of lysate were kept as input and 900 μl were incubated with 1 μg of anti-TRF1 antibody (St John’s laboratory) for 4 h at 4 °C.

Techniques: Western Blot, Transduction, Plasmid Preparation, Two Tailed Test, MANN-WHITNEY, Two-Dimensional Gel Electrophoresis, Migration, Over Expression, Control

Journal: Nature Communications

Article Title: TRF1 relies on fork reversal to prevent fragility at human telomeres

doi: 10.1038/s41467-025-61828-5

Figure Lengend Snippet: A Lagging (green) and leading (black) telomere fragility assessed by CO-FISH in PCS LT -EV, PCS LT -TRF1 or PCS LT -TRF1-RH1 cells under the specified conditions. siSM1: siSMARCAL1. Cells were treated with 60 µM spironolactone (SP), or DMSO as control, during 25 h before BrdU/BrdC incorporation. A minimum of 5900 chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Kruskal–Wallis tests. Source data are provided as a Source Data file. B Model . Stalled replication forks at PCS LT telomeres undergo SMARCAL1-dependent reversal, generating new 3′ telomeric ends. The reversed forks are bound by telomerase. Fork replication can resume through a homologous recombination-dependent restart process that requires RAD51 and might be facilitated by long 3′ overhangs generated by the telomerase and the formation of RNA:DNA hybrid formation, possibly involving TFIIH. If reversal is suppressed, PrimPol-dependent repriming can promote fork restart. This process may also be dependent on RNA:DNA hybrids. Optimal levels of TRF1 are required to suppress telomere fragility, by facilitating the reversal/restart processing of stalled forks. Protein illustrations were generated using BioRender (Vaurs, M. (2025) https://BioRender.com/ke04c93 ).

Article Snippet: One hundred microliters of lysate were kept as input and 900 μl were incubated with 1 μg of anti-TRF1 antibody (St John’s laboratory) for 4 h at 4 °C.

Techniques: Control, Homologous Recombination, Generated

Journal: Nature structural & molecular biology

Article Title: Deregulated DNA ADP-ribosylation impairs telomere replication.

doi: 10.1038/s41594-024-01279-6

Figure Lengend Snippet: Fig. 3 | Direct targeting of telomeres via a DarT-TRF1 endotoxin fusion. a, Schematic of GFP-tagged DarT-TRF1-induced telomeric DNA ADP-ribosylation and subsequent removal by DarG (red). b, Immunofluorescence of U2OS CTRL and TARG1-KO cells after expression of GFP-tagged WT-DarT-TRF1 (WT) or E160A DarT-TRF1 (mut) and FLAG-tagged WT-DarG or K80A DarG (left) and quantification of colocalization of mono-ADPr and GFP-positive telomeres (right). In all conditions, more than 140 cells were analyzed. Scale bars represent 10 μm. c, Western blot of U2OS CTRL and TARG1-KO cells after expression of GFP- tagged WT-DarT-TRF1 (WT) or E160A DarT-TRF1 (mut) and FLAG-tagged WT-DarG

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging 3 nature portfolio | reporting sum m ary April 2023 Antibodies Antibodies used Immunofluorescence: FLAG (D6W5B) (#14793S, Cell Signaling Technology, 1:1000); FLAG (M2) (#F1804, Millipore, 1:1000); TRF1 (#ab10579, Abcam, 1:1000); TRF2 (#NB110-57130, Novus, 1:1000); RPA2 (#ab2175 Lot #GR3386072-1, Abcam, 1:1000), Poly/MonoADP-ribose (#83732, Lot #5, Cell Signaling Technology, 1:1000); Mono-ADP-ribose (#HCA354 Lot #162452, Biorad, 1:1000); MonoADP-ribose (#HCA355 Lot #158442, Biorad, 1:1000); Cas9 (Cat. #14697 Lot #8, Cell Signaling Technology, 1:1000); GFP #ab6556, Abcam, 1:1000); Alexa Fluor 594 goat anti-rabbit (#A11012 Lot #2433881, Thermo Fisher, 1:1000); Alexa Fluor 594 goat anti-mouse. (#A11032 Lot #2527968, Thermo Fisher, 1:1000); Alexa Fluor 488 goat anti-mouse (#A11001 Lot #2659299, Thermo Fisher, 1:1000); Alexa Fluor 488 goat anti-rabbit. (#A32731 Lot #XD343356, Thermo Fisher, 1:1000); Alexa Fluor 647 goat anti-mouse. (#A21244 Lot #2527970, Thermo Fisher, 1:1000).

Techniques: Immunofluorescence, Expressing, Western Blot

Journal: Diagnostics

Article Title: Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD

doi: 10.3390/diagnostics12020393

Figure Lengend Snippet: Oligonucleotides used for cloning and sequencing.

Article Snippet: The recombinant ACE-2, RBD and beta-RBD proteins described in this study is available under a standard MTA with the Federal Research Center “Fundamentals of Biotechnology” Russian Academy of Sciences. pTM, pTM-RBDv2, pTF, pTF-ACE2s are available from Addgene: #162783; #162785; #162784; 162786.

Techniques: Cloning, Sequencing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The 68-kDa Telomeric Repeat Binding Factor 1 (TRF1)-associated Protein (TAP68) Interacts with and Recruits TRF1 to the Spindle Pole during Mitosis *

doi: 10.1074/jbc.M113.526244

Figure Lengend Snippet: Identification of a novel TRF1-binding protein, TAP68. A, mitotic HeLa cell extracts were applied to a TRF1 peptide antibody affinity column. After binding, columns were extensively washed, and bound proteins were eluted and separated by SDS-PAGE. The indicated proteins were extracted from the gel and digested with trypsin, and the amino acid sequence of the peptides was determined by MALDI-TOF and LC-MS/MS mass spectrometry. P1 is TAP68. B, protein band of P1 was extracted from the acrylamide gel and digested with trypsin, and the resulting peptide fragments were subjected to mass spectrometric analysis. C, P1 peptides match a previously uncharacterized protein with a 593-amino acid open reading frame of unknown function (AAH03618). D, schematic representation of the TAP68 domain organization predicted by NCBI's on-line conserved domain search tool. E, TRF1-TAP68 interaction was confirmed by immunoprecipitation assay. The anti-TRF1 antibody binding on protein A/G beads was incubated with mitotic cell lysate prepared in high stringency lysis buffer containing 1% Nonidet P-40 and 0.5% deoxycholate. Under this condition, TAP68 remained firmly bound to TRF1, whereas the majority of other TRF1-binding proteins, such as TRF2 and tankyrase, were absent from the co-immunoprecipitates.

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Binding Assay, Affinity Column, SDS Page, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Acrylamide Gel Assay, Immunoprecipitation, Incubation, Lysis

Journal: The Journal of Biological Chemistry

Article Title: The 68-kDa Telomeric Repeat Binding Factor 1 (TRF1)-associated Protein (TAP68) Interacts with and Recruits TRF1 to the Spindle Pole during Mitosis *

doi: 10.1074/jbc.M113.526244

Figure Lengend Snippet: Biochemical characterization of TRF1-TAP68 interaction. A, exogenous TRF1 interacts with TAP68 in HeLa cells transfected with FLAG-tagged TRF1 and either GFP vector or GFP-tagged TAP68. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and FLAG-tagged TRF1 protein and its binding proteins were immunoprecipitated (IP) with 20 μl of FLAG-M2 antibody-conjugated beads. Co-precipitated proteins were separated by SDS-PAGE and then immunoblotted for the presence of FLAG-TRF1 (upper panel) or GFP-TAP68 proteins (lower panel). B, exogenous TAP68 interacts with TRF1 in HeLa cells transfected with FLAG-tagged TRF1 and either GFP or GFP-tagged TAP68. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and GFP-tagged TAP68 protein and its associated proteins were precipitated with 20 μl of GFP antibody-conjugated beads. Co-precipitated proteins were then detected by immunoblot for the presence of GFP-TAP68 (upper panel) or FLAG-TRF1 proteins (lower panel). C, schematic representation of different TAP68 truncation constructs. D, HeLa cells were transfected with full-length or different truncations of GFP-tagged TAP68 along with FLAG-tagged TRF1. After 36 h, HeLa cells were then extracted with 1% Nonidet P-40 plus 0.5% deoxycholate, and FLAG-tagged TRF1 and its accessory proteins were precipitated with 20 μl of rabbit TRF1 antibody-conjugated beads. Co-precipitated proteins were then immunoblotted for the presence of GFP-TAP68 proteins (upper panel) or FLAG-TRF1 (lower panel).

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Transfection, Plasmid Preparation, Binding Assay, Immunoprecipitation, SDS Page, Western Blot, Construct

Journal: The Journal of Biological Chemistry

Article Title: The 68-kDa Telomeric Repeat Binding Factor 1 (TRF1)-associated Protein (TAP68) Interacts with and Recruits TRF1 to the Spindle Pole during Mitosis *

doi: 10.1074/jbc.M113.526244

Figure Lengend Snippet: TAP68 mediates TRF1 localization to the centrosome. A, TAP68 and TRF1 share similar subcellular localization. Nuclear and post-nuclear fractions were generated from interphase HeLa cell homogenates. Equal amounts of proteins (35 μg) from various fractions were separated by 5–15% gradient SDS-PAGE and detected by Western blot analyses using antibodies against TAP68, TRF1, and β-tubulin, respectively. B, representative images of interphase (top panels), late prophase/early prometaphase (middle panels), and metaphase (bottom panels) HeLa cells stained for TRF1 (green), TAP68 (red), DAPI (blue), and γ-tubulin (white). In interphase cells, TRF1 (panel a) and TAP68 (panel b) are readily seen in the nucleus as speckles and dots in the merge panel (panel d). In late prophase or the earliest prometaphase cells marked by nuclear membrane fragmentation, TAP68 appears at the centrosome (panel f, arrows), and TRF1 remains as speckles in the partially condensed chromosome (panel e, arrows). Both TAP68 and TRF1 become co-localized to the centrosome as the chromosomes align or after sister chromatid separation (panels i and j, arrows). A merged image shows the co-localization of TRF1 and TAP68 to the centrosome of mitotic cells (panel l). Bars, 10 μm. C, immunoelectron microscopy of late prophase cells indicated that TAP68 localizes to the pericentrioles. D, representative immunofluorescence images of HeLa cells treated with nocodazole or DMSO. 10 h after drug treatment, the cells were fixed and stained for tubulin (green), TAP68 (red), and DAPI (blue). TAP68 remained centrosome-associated in the absence of microtubules (panel h, arrows). E, ectopic expression of different GFP-tagged TAP68 proteins. HeLa cells were transfected GFP-tagged full-length TAP68 or its deletion mutants. After 36 h, HeLa cells were then harvested for Western blot analysis using a GFP antibody. F, representative images collected from metaphase HeLa cells transiently transfected with GFP-tagged TAP68 and its deletion mutants and stained for TRF1 (red), TAP68 (green), and DAPI (blue). In cells expressing full-length TAP68, both TAP68 and TRF1 are co-localized to the centrosome (panels a, b, and d, arrows). A similar co-distribution of TRF1 with TAP68 was observed in TAP68ΔC mutant-expressing cells (panels e, f, and h) but not TAP68MC-expressing cells (panels i, j, and l), demonstrating that the centrosomal localization region of TAP68 is independent of its TRF1-binding domain. Bars, 10 μm. G, schematic representation of the regions of TAP68, which specifies its centrosomal localization. H, representative images of HeLa cells transfected with different GFP-tagged TAP68 constructs. 24 h after transfection, cells were fix and co-stained for γ-tubulin (red) and DNA (blue). Bars, 10 μm.

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Generated, SDS Page, Western Blot, Staining, Membrane, Immuno-Electron Microscopy, Immunofluorescence, Expressing, Transfection, Mutagenesis, Binding Assay, Construct

Journal: The Journal of Biological Chemistry

Article Title: The 68-kDa Telomeric Repeat Binding Factor 1 (TRF1)-associated Protein (TAP68) Interacts with and Recruits TRF1 to the Spindle Pole during Mitosis *

doi: 10.1074/jbc.M113.526244

Figure Lengend Snippet: TAP68 mediates TRF1 translocation to the centrosome and mitotic regulation. A, efficient siRNA-mediated suppression of TAP68. HeLa cells were transfected with a siRNA duplex specific for TAP68 and a scrambled control siRNA duplex. After 48 h, HeLa cells were then harvested for Western blot analysis using antibodies specific for TAP68, TRF1, tankyrase 1 (TANKS1), and NuMA. The analyses indicate the specificity and efficiency of the targeted protein suppression. B, representative immunofluorescence images of HeLa cells transfected with control or TAP68-specific siRNA duplex. 48 h post-transfection, cells were stained for the indicated antibodies and DNA, respectively. In the control duplex-transfected cells, NuMA, TAP68, and tankyrase 1 are all co-localized to the centrosome (panels a–c). In TAP68 siRNA duplex-treated cells, TAP68 protein expression was suppressed (panels f and j), and localization of TRF1 to the centrosome was diminished, although TRF1 distribution on chromosome remained (panel g). The distribution of tankyrase 1 to the centrosome was also eliminated in TAP68-depleted cells (panel k). However, the spindle pole localization of NuMA was not altered by the loss of TAP68 (panel e). Tetrapolar spindle in TAP68-depleted cells was revealed by NuMA staining (panel e). Bars, 10 μm. C, quantitative analyses of the mitotic spindle after TAP68 knockdown. HeLa cells were transfected with TAP68 siRNA oligonucleotide and control oligonucleotide for 36–48 h followed by fixation and staining for DNA, TAP68, and NuMA. After examination under a fluorescence microscope, the centrosome instability phenotypes were categorized as bipolar, tripolar, or tetrapolar spindles. Data represent the percentage of total mitotic cells evaluated. An average of 100 cells from three separate experiments was counted. * indicates a significant difference between the TAP68 siRNA- and control siRNA-transfected cells.

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Translocation Assay, Transfection, Control, Western Blot, Immunofluorescence, Staining, Expressing, Knockdown, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: The 68-kDa Telomeric Repeat Binding Factor 1 (TRF1)-associated Protein (TAP68) Interacts with and Recruits TRF1 to the Spindle Pole during Mitosis *

doi: 10.1074/jbc.M113.526244

Figure Lengend Snippet: Phospho-regulation of TRF1-TAP68 interaction and centrosomal localization during mitosis. A, immunoblot analysis of TAP68 in HeLa cells synchronized at the indicated cell cycle phases. Note the mobility shift (pTAP68) from mitotic cell lysates. B, HeLa cell lysates (35 μg) from synchronized interphase and mitotic cells were treated with λ-phosphatase or the phosphatase inhibitor, okadaic acid, followed by Western blotting with an anti-TAP68 antibody. C, bacterially expressed histidine-tagged TAP68 fusion proteins, both wild-type and mutant (TAP68T221A and TAP68T457A), were phosphorylated in vitro in the presence of [32P]ATP and NEK2A kinase. Samples were separated by 6–16% gradient SDS-PAGE. The gel was dried and subsequently incubated with x-ray film. Note that in the presence of NEK2A, there was a dramatic incorporation of 32P into the wild-type TAP68 and TAP68T457A mutant, but not the TAP68T221A mutant. D, purified His-tagged TAP68-WT and TAP68-T457A were subjected to in vitro phosphorylation by recombinant PLK1 kinase. The left panel shows Coomassie Blue staining of the gel. The right panel shows the result of autoradiography. E, HeLa cell lysates from synchronized G1 and nocodazole-treated mitotic cells ectopically expressing wild-type or the indicated FLAG-TAP68 mutants were immunoblotted with an anti-FLAG and anti-α-tubulin antibodies, respectively. F, lysates from mitotic HeLa cells ectopically expressing FLAG-tagged TAP68-WT and the indicated mutants, immunoprecipitated with an anti-TRF1 antibody, and then immunoblotted with anti-TRF1 and anti-FLAG antibodies. TAP68T221A, which cannot be phosphorylated by NEK2A, was co-precipitated with TRF1 (lane 4), and its phospho-mimicking mutant TAP68T221E did not interact with TRF1 (lane 6). In contrast, the mutant with mimics Thr-457 phosphorylation by PLK1, TAP68T457E, interacted with TRF1, whereas the TAP68T457A mutant did not (lanes 10 and 8, respectively). Thus, phosphorylation of TAP68 at Thr-221 by NEK2A releases it from TRF1, whereas phosphorylation at Thr-457 by PLK1 is required for the TRF1-TAP68 interaction. G, representative images of HeLa cells ectopically expressing wild-type and mutant FLAG-TAP68. Cells were fixed and stained for TAP68 (red), TRF1 (green), and DNA (blue). Bars, 10 μm. Both wild-type FLAG-TAP68 and TRF1 are co-localized to the centrosome at metaphase (panels a and b, arrows). A merged image shows the co-localization of TRF1 and wild-type FLAG-TAP68 to the centrosome of mitotic cells (panel d). However, in HeLa cells ectopically expressing TAP68T221A, which cannot be phosphorylated by NEK2A, TAP68T221A remains associated with TRF1 at the telomere in late prometaphase cells (panel f) as evident in the merged image (panel h). In HeLa cells ectopically expressing TAP68T457A, which cannot be phosphorylated by PLK1, TAP68T457A localizes to the centrosome (panel j) with little TRF1 (panel i) as evidenced in the merged image (panel l, arrows), illustrating that PLK1-mediated phosphorylation is essential for TAP68-TRF1 association at the centrosome. Indeed, TRF1 co-localizes with TAP68 in HeLa cells ectopically expressing the phospho-mimicking mutant, TAP68T457E.

Article Snippet: Mitotic extracts from HeLa S3 cells (1 × 10 9 ) were prepared in lysis buffer (25 m m HEPES, pH 6.9, 150 m m NaCl, 2 m m EGTA, 5 m m MgCl 2 , 0.2% Triton X-100, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml chymostatin, and 10 μg/ml pepstatin A) and incubated with 100 μl of protein A-Sepharose beads coupled with a rabbit anti-TRF1 antibody (Ab13792; Novus Biologicals, Inc.) using a previously described protocol ( 24 ).

Techniques: Western Blot, Mobility Shift, Mutagenesis, In Vitro, SDS Page, Incubation, Purification, Phospho-proteomics, Recombinant, Staining, Autoradiography, Expressing, Immunoprecipitation