Journal: Nature structural & molecular biology

Article Title: The macroH2A1.2 histone variant links ATRX loss to alternative telomere lengthening.

doi: 10.1038/s41594-019-0192-3

Figure Lengend Snippet: (A) ChIP for γ-H2AX at the indicated loci in K562 cells in the presence (WT) or absence of macroH2A1.2 (1.2 CRISPR-KO). Samples were normalized to untreated WT samples, values represent mean and s.d. from 3 independent experiments. See for macroH2A1.2 ChIP. (B) γ-H2AX ChIP at the indicated loci in U2OS cells treated with vehicle (DMSO) or HU in the presence or absence of ATRX re-expression. Values represent mean and s.d. from 3 independent experiments. (C) Frequency of TIFs in U2OS cells with ATRX induction in the presence or absence of Aph, TIFs were defined based on co-localization of γ-H2AX (red) and TRF2 (green), a representative image from Aph-treated, sh-RFP expressing U2OS cells is shown; scale bar = 5 μm. Box plots depict the number of TIFs per cell. N: number of cells. *** p < 10 −8 by Mann-Whitney U test, one of two independent experiments is shown. (D) Model linking ATRX and macroH2A1.2 to ALT telomere maintenance. ATRX-dependent macroH2A1.2 retention at stalled replication forks protects from excessive DNA damage in ALT-negative cells. In ALT-positive cells, ATRX deficiency leads to macroH2A1.2 loss and DSBs in response to RS, which triggers DDR-dependent macroH2A1.2 re-deposition to facilitate HR. In the absence of RS, lack of ATRX has little effect on telomeric macroH2A1.2 levels, pointing to DNA damage-induced modulation of ATRX function, which may involve ATRX phosphorylation in S phase (see ) .

Article Snippet: The following antibodies were used for ChIP: α-macroH2A1.2 (Millipore MABE61), α-phospho-S139-H2AX (Millipore 05–636), α-H2B (Abcam ab52484), α-H2A (Abcam ab18255), TRF2 (Novus Biologicals IMG-124A) and normal mouse IgG (Millipore 12–371).

Techniques: CRISPR, Expressing, MANN-WHITNEY

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: Journal of Biological Chemistry

Article Title: Androgen Receptor Interacts with Telomeric Proteins in Prostate Cancer Cells

doi: 10.1074/jbc.m109.098798

Figure Lengend Snippet: FIGURE 1. Casodex disrupts telomeric complexes in AR-positive prostate cancer cells. A and B, LNCaP cells treated with 100 M Casodex for 48 h (A and B) or with 20 g/ml etoposide for 1 h (B) were co-immunostained with antibodies against 53BP1 and TRF2. LNCaP (C) or PC-3 (D) cells were treated with or without 100 M Casodex for 48 h and then immunostained with 53BP1 antibody. 53BP1 foci were counted, and data are presented as the percentage of cells with 0–5, 6–10, 11–20, or 20 foci/cell. Immunostaining and confocal microscopy were performed as described under “Experimental Procedures.” 80 cells in each treatment group were scored in three separate experiments.

Article Snippet: For Western blot analysis, membraneswere probedwith antibodies against AR (AR-N20, Santa Cruz Biotechnology), TRF2 (IMG-124A), TRF1 (H-242, Santa Cruz Biotechnology), HA (sc-805, Santa Cruz Biotechnology), TIN2 (15), TPP1 (Abcam), or glyceraldehyde-3-phosphate dehydrogenase (Chemicon).

Techniques: Immunostaining, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: Androgen Receptor Interacts with Telomeric Proteins in Prostate Cancer Cells

doi: 10.1074/jbc.m109.098798

Figure Lengend Snippet: FIGURE 2. Casodex disrupts telomeric complexes without affecting the expression of telomeric proteins. A, LNCaP cells treated with 100 M Casodex for 0, 3, 10, 24, or 48 h were immunostained for 53BP1. 53BP1 foci were counted, and cells were categorized as having 5 or 5 foci/cell. 200 cells in each treatment group were scored in three independent experiments. B, total RNA from LNCaP cells treated with Casodex was extracted using TRIzol (Invitrogen), and RT-PCR was performed as described previously (32) to measure mRNA levels. Sequence-specific primers for TIN2, TRF1, TRF2, TPP1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (11), and for AR, 5-tcagttcacttttgacctgctaa-3 (forward) and 5-gtggaaatagat- gggcttga-3 (reverse) primers were used. C, whole cell lysates of LNCaP cells treated with Casodex were sub- jected to Western blot analysis to determine TIN2, TRF1, TRF2, AR, and glyceraldehyde-3-phosphate dehydro- genase (loading control) protein levels.

Article Snippet: For Western blot analysis, membraneswere probedwith antibodies against AR (AR-N20, Santa Cruz Biotechnology), TRF2 (IMG-124A), TRF1 (H-242, Santa Cruz Biotechnology), HA (sc-805, Santa Cruz Biotechnology), TIN2 (15), TPP1 (Abcam), or glyceraldehyde-3-phosphate dehydrogenase (Chemicon).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Androgen Receptor Interacts with Telomeric Proteins in Prostate Cancer Cells

doi: 10.1074/jbc.m109.098798

Figure Lengend Snippet: FIGURE 4. AR is associated with telomeric proteins in LNCaP cells. A, telomeric proteins are overexpressed in prostate cancer cells. Whole cell lysates prepared from prostate cancer cells (LNCaP, PPC-1, and PC-3) and normal prostate epithelial cells (PrEC) were subjected to Western blot analysis to determine TRF1, TRF2, TIN2, AR, and -actin (loading control) protein levels. B, AR and TRF2 in LNCaP cell lysates were individually immunoprecipitated (IP) using monoclonal antibodies, and immunopre- cipitates and starting lysate (2% of input) were subjected to Western blot (WB) analysis of AR, TRF1, and TRF2. C, cell lysates were prepared from LNCaP cells transfected with HA-tagged TRF1 as described previously (13), and HA-TRF1 in cell lysates was immunoprecipitated using antibodies against the HA epitope. Immunoprecipitates and unprecipitated lysate (10% of input) were subjected to Western blot analysis of AR and TIN2. D, sucrose density gradient analysis of telomeric proteins and AR in LNCaP cells. Nuclear extract (NE) prepared from exponentially growing LNCaP cells was subjected to sucrose density gradient centrifugation, and the gradient was resolved into 12 fractions, which were subjected to Western blot analysis to identify the distribution of TRF1, TRF2, TIN2, TPP1, and AR in the gradient. Lamin B was used as a control. Top, top of the gradient; Bottom, bottom of the gradient; NE, Nuclear extract loaded onto the gradient.

Article Snippet: For Western blot analysis, membraneswere probedwith antibodies against AR (AR-N20, Santa Cruz Biotechnology), TRF2 (IMG-124A), TRF1 (H-242, Santa Cruz Biotechnology), HA (sc-805, Santa Cruz Biotechnology), TIN2 (15), TPP1 (Abcam), or glyceraldehyde-3-phosphate dehydrogenase (Chemicon).

Techniques: Western Blot, Control, Immunoprecipitation, Bioprocessing, Transfection, Gradient Centrifugation

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. Equimolar amounts of GST, GST-ELK and His-TRF2 were incubated in the absence (−) or presence (+) of recombinant active ERK2. The phosphorylated proteins were detected after SDS PAGE using a specific anti-PX[phospho]SP antibody (pPXSP). Coomassie blue staining of the membrane is shown as a loading control. B. Alignment of TRF2 sequences from different mammalian species show the conservation of a MAPK phosphorylation consensus PXSP target site. The species and respective Genbank reference numbers corresponding to the sequences are reported. The conserved PXSP site is shown in bold and the conserved S residue underlined. C. Specificity of the immune serum using peptides containing phospho-S323. His-TRF2 was phosphorylated or not by recombinant active ERK2. The same amounts of proteins were submitted to immunoblotting analysis with the anti-pS323 antibody (pTRF2). Coomassie blue staining is shown as a loading control. D. A375 cells were stably transfected with WT-TRF2 or TRF2 S323A . Cells were treated (+) or not (−) with PD184352 (PD). Phosphorylated TRF2 was immunoprecipitated with the specific anti-pTRF2 antibody and detected by immunoblotting with an anti-TRF2 antibody (IP). Total TRF2 is shown as a loading control (input) and pERK1/2 as a control of PD184352 activity.

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Incubation, Recombinant, SDS Page, Staining, Membrane, Control, Phospho-proteomics, Residue, Western Blot, Stable Transfection, Transfection, Immunoprecipitation, Activity Assay

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. Immortalized BJ fibroblasts were serum deprived for 24 hours. BJ cells were then stimulated with 10% FCS for the indicated times. Phosphorylated forms of TRF2 were immunoprecipitated with the specific anti-pTRF2 antibodies and detected by immunoblotting with an anti-TRF2 antibody (IP p-TRF2). Total TRF2 and Hsp-90 are shown as loading controls and the phosphorylated forms of ERK1/2 as a control of serum-dependent activation of ERK1/2 (Input). B. Different tumor cells were tested for the presence of pTRF2 by immunoprecipitation in the presence (+) or absence (−) of PD184352 (PD) (Cal33, BJ-Ras (BJ-R), A375, U2OS). A short (Short exp.) and long (Long exp.) exposure of the blots are shown (IP p-TRF2). Total TRF2, Hsp90 and tubulin are shown as loading controls and the phosphorylated forms of ERK1/2 as a control of PD184352 activity (Input).

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Immunoprecipitation, Western Blot, Control, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. In situ proximity ligation assay (PLA) with anti-TRF2 and anti-pERK1/2 antibodies alone (negative controls, but in the presence of the two secondary antibodies) or in combination (TRF2 + pERK1/2) in A375 cells in the presence of DMSO or PD184352 (scale bars represent 10μm). A 3D image reconstruction after confocal microscopy imaging of PLA with a combination of anti-TRF2 and anti-pERK1/2 antibodies is also shown (scale bar represents 1μm). B. PLA with combined anti-TRF2 and anti-pERK antibodies in normal human skin tissue or cutaneous squamous cell carcinoma, normal lung or lung squamous cell carcinoma and normal cervix or cervical squamous cell carcinoma (scale bars represent 35μm). A 3D image reconstruction after confocal microscopy imaging of PLA with a combination of anti-TRF2 and anti-pERK1/2 antibodies in cutaneous squamous cell carcinoma is also shown (scale bar represents 1μm).

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: In Situ, Proximity Ligation Assay, Confocal Microscopy, Imaging

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. A375, SKMel-51 and BJ-RAS cells were incubated in the presence of 50 μg/ml cycloheximide (CHX) for 16 hours in the presence or absence of PD184352 (PD). Total TRF2 and tubulin amounts were evaluated by immune-blotting. Tubulin and coomassie blue staining of the membrane (B) are shown as loading controls. B. Densitometric quantifications of the blots shown in A. The TRF2 expression level was normalized with three to four different loading controls and expressed relative to the control conditions (results are expressed as mean ± SD). One way ANOVA statistical analysis is included: * p<0.05; ** p<0.01; *** p<0.001). C. A375 cells over-expressing either TRF2-WT or TRF2-S323A were incubated in the presence of 50 μg/ml cycloheximide for 16 hours. Tubulin is shown as loading control. Densitometric quantification of the blot is shown (results are expressed as mean ± SD. One way ANOVA statistical analysis is included: * p<0.05; ** p<0.01; *** p<0.001).

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Incubation, Staining, Membrane, Expressing, Control

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. Confocal imaging of the co-staining of 53BP1 by immunofluorescence (red) and telomeres by fluorescent in situ hybridization (TelC-FITC, green) in A375 cells were the expression of WT-TRF2, TRF2 S323A or TRF2-ΔBΔM was induced by tetracycline (Tet) treatment. Colocalisation events were counted as Telomere dysfunction-Induced Foci (TIF, indicated with white arrows, scale bars represent 5μM) and the proportion of nuclei showing more than 3 TIF is indicated (right panel, results are expressed as mean ± SD, t-test statistical analysis is included: * p<0.05; ** p<0.01; *** p<0.001). B. The proportion of cells in each phase of the cell cycle was determined by DNA labeling with propidium iodide and FACS analysis. Sub G1 stands for cells with fragmented DNA, a hallmark of apoptosis. C. Conditional overexpression of different forms of TRF2 (WT, TRF2 S323A and TRF2 ΔBΔM ) was induced by tetracycline (Tet) in A375 cells. Seven days after tetracycline stimulation, cells were colored with giemsa blue. A close-up in TRF2 S323A overexpression well shows the few remaining cells at the end of the experiment. D. The cells were tested for b-galactosidase activity after seven days of tetracyclin induction (lower pictures). The percentage of β-galactosidase positive cells under tetracycline-induced conditions is specified below the images. E. Seven days after induction of the different forms of TRF2 by tetracycline (+), cells were tested for the presence of phosphorylated forms of p53. Actin and p53 are shown as loading controls.

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Imaging, Staining, Immunofluorescence, In Situ Hybridization, Expressing, DNA Labeling, Over Expression, Activity Assay

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. Control A375 cells or A375 cells conditionally expressing WT-TRF2 or TRF2 S323A were subcutaneously injected into nude mice. Doxycycline was added to the drinking water ten days after injection to induce the transgenes expression. The tumor volume is shown and results are expressed as mean ± SD (t-test statistical analysis is included: * p<0.05; ** p<0.01; *** p<0.001). B. Immunoblots showing the expression of TRF2 and actin (loading control) in tumor extracts prepared at the end of the tumor xenograft experiment.

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Control, Expressing, Injection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: hSnm1B Is a Novel Telomere-associated Protein

doi: 10.1074/jbc.c600038200

Figure Lengend Snippet: FIGURE1.hSnm1B,butnothSnm1A,localizestothetelomere.293TcellswereplatedoncoverslipsandtransientlytransfectedwitheitherYFP-FLAG-hSnm1A(left)orGFP-hSnm1B (right). The cells were then fixed, permeabilized, and incubated with TRF2 monoclonal antibody. The TRF2 antibody was visualized with the rhodamine (TRITC)-conjugated donkey anti-mouseIgGantibody(top).YFP-FLAG-hSnm1Awasfoundtolocalizetopunctatenuclearbodies(centerleft)butdidnotco-localizewithTRF2(bottomleft).GFP-hSnm1Bwasfound to localize to punctate nuclear bodies (center right) that co-localize with TRF2 (bottom right).

Article Snippet: To visualize TRF2, 293T cells were then permeabilized with 0.5% Nonidet P-40 in 1 PBS and incubated with the anti-human TRF2 monoclonal antibody (Imgenex IMG-124A) at 1:5000 dilution.

Techniques: Incubation

Journal: Journal of Biological Chemistry

Article Title: hSnm1B Is a Novel Telomere-associated Protein

doi: 10.1074/jbc.c600038200

Figure Lengend Snippet: FIGURE3.TRF2interactswiththeCterminusofhSnm1B.A,293Tcellsweretransiently transfected with GFP-hSnm1B and immunoprecipitated with GFP monoclonal antibody. The resultant immunoprecipitates were separated on a polyacrylamide gel and blotted with either GFP monoclonal antibody (top) or TRF2 monoclonal antibody (bottom). GFP- hSnm1B was found to co-immunoprecipitate with endogenous TRF2. Control mock- transfected cells were unable to co-immunoprecipitate TRF2. B, to elucidate the TRF2- binding domain, the indicated GFP-tagged hSnm1B truncation mutants were generated. C, 293T cells were transiently transfected with YFP-FLAG-hSnm1A, GFP- hSnm1B, or a GFP-tagged truncation mutant of hSnm1B, followed by immunoprecipita- tion with GFP monoclonal antibody, separation on a polyacrylamide gel, and blotting with either GFP polyclonal antibody (top) or TRF2 monoclonal antibody (bottom). All of these mutants were able to co-immunoprecipitate endogenous TRF2 except mutant GFP-hSnm1B (363–495). YFP-FLAG-hSnm1A was unable to co-immunoprecipitate TRF2.

Article Snippet: To visualize TRF2, 293T cells were then permeabilized with 0.5% Nonidet P-40 in 1 PBS and incubated with the anti-human TRF2 monoclonal antibody (Imgenex IMG-124A) at 1:5000 dilution.

Techniques: Transfection, Immunoprecipitation, Control, Binding Assay, Generated, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: hSnm1B Is a Novel Telomere-associated Protein

doi: 10.1074/jbc.c600038200

Figure Lengend Snippet: FIGURE 4. The TRF2 homology domain is required for interaction with hSnm1B. A, to elucidate the hSnm1B interaction domain on TRF2, the indicated N-terminal myc epitope-tagged TRF2 truncation mutants were generated. B, 293T cells were transiently transfected with GFP-hSnm1B and either myc-TRF2 or a myc-tagged TRF2 truncation mutant, followed by immunoprecipitation with GFP monoclonal antibody. The immu- noprecipitates were then separated on a polyacrylamide gel and blotted with either GFP monoclonal antibody (top) or myc antibody (middle). 5% of the nuclear extracts were used for inputs to verify expression of the TRF2 truncation mutants (bottom). hSnm1B was found to strongly interact only with wild-type TRF2 and the region of TRF2 contain- ing the complete TRF2 homology domain.

Article Snippet: To visualize TRF2, 293T cells were then permeabilized with 0.5% Nonidet P-40 in 1 PBS and incubated with the anti-human TRF2 monoclonal antibody (Imgenex IMG-124A) at 1:5000 dilution.

Techniques: Generated, Transfection, Mutagenesis, Immunoprecipitation, Expressing