chip assays include rabbit polyclonal antibodies orc2  (Bethyl)

Journal: Cell reports

Article Title: TRF2 Mediates Replication Initiation within Human Telomeres to Prevent Telomere Dysfunction

doi: 10.1016/j.celrep.2020.108379

Figure Lengend Snippet: (A) Doxycycline (Dox)-inducible expression of Myc-tagged TRF2ΔB. rtTA, reverse tetracycline trans -activator; TRE, tetracycline response element. (B) Immunoblot of cell lysates of LOX cell line stably expressing Dox-inducible TRF2ΔB. Untagged endogenous WT TRF2 (upper band) and Myc-tagged TRF2ΔB (lower band) are indicated. (C) ChIP analysis of LOX TRF2ΔB cells grown in the absence (−) or presence (+) of 1 μg/mL Dox for 4 days using antibodies against TRF2, ORC2, MCM3, H3K9me3, or immunoglobulin G (IgG) (control). Blots were probed by hybridization with 32 P-labeled telomeric TTAGGG (TelG) or Alu repeat (Alu) probes. Long Exp, long exposure. (D) Quantification of at least three independent experiments represented in (C). The ChIP data were first normalized to input, and the percent input for Dox (+) is shown as relative to dox (−), which was set as 1. Student’s t test was used for statistical analysis. Error bars indicate SD. **p < 0.01, ***p < 0.001; ns, no statistical significance (p > 0.05). (E) SMARD analysis of the Ch7q telomere segment from LOX TRF2ΔB cells grown for 3 days in the presence of 1 μg/mL Dox. Alignments of replicated molecules fully labeled with IdU (red) and CldU (green) are shown, collected from six independent samples stretched on slides (76 fully red- and 79 fully green-labeled molecules were also collected). Vertical lines (orange and blue) demarcate the boundaries where FISH probes bind, as described in . Symbols are as in . A replication profile histogram is shown under the molecule alignment.

Article Snippet: Antibodies used in ChIP assays include rabbit polyclonal antibodies ORC2 (Bethyl), MCM3 (Abcam), histone H3K9me3 (Diagenode) or IgG (Cell Signaling).

Techniques: Expressing, Western Blot, Stable Transfection, Control, Hybridization, Labeling

Journal: Cell reports

Article Title: TRF2 Mediates Replication Initiation within Human Telomeres to Prevent Telomere Dysfunction

doi: 10.1016/j.celrep.2020.108379

Figure Lengend Snippet: (A) Immunoblot of cell lysates of LOX cell lines stably expressing Dox-inducible SNF2H shRNA or Luc control shRNA. (B) ChIP analysis of LOX cells stably expressing Dox-inducible SNF2H shRNA grown in the absence+ (−) or presence (+) of 1 μg/mL Dox for 10 days using antibodies against ORC2, MCM3, or IgG (control). Blots were probed by hybridization with 32 P-labeled TelG or Alu probes. (C) Quantification of four independent experiments represented in (B). The ChIP data were first normalized to input, and the percent input for Dox (+) is shown as relative to Dox (−), which was set as 1. Student’s t test was used for statistical analysis. Error bars indicate SD. **p < 0.002; ns, p > 0.05. (D) Telomere length analysis of LOX cell lines stably expressing Dox-inducible SNF2H shRNA or Luc control shRNA. Cells were grown in the absence (−) or presence (+) of 1 μg/mL Dox for 10 days. Telomere length and relative amount of telomeric DNA were determined by restriction digestion of genomic DNA with AluI/MboI, followed by PFGE and Southern hybridization with a 32 P-labeled (CCCTAA) 4 probe. Ethidium bromide staining of the total DNA digest was used to normalize for DNA loading (bottom). Fragment size (in kilobases) is indicated on the left of the blot. The relative intensity of telomeric DNA signals (− Dox versus + Dox) is indicated below the blot. (E) Model of telomeric origin function in telomere replication. The repetitive sequence of telomeric DNA impedes and stalls replication forks. Failure to restart replication (i) leads to incomplete replication, telomere repeat loss, and dysfunction. Restart of replication from telomeric origins (ii) results in completion of telomere replication and proper telomere maintenance. (F) Model of telomeric origin assembly and firing. (i) SNF2H remodels telomere chromatin to allow ORC loading. (ii) TRF2 recruits the ORC. (iii) The ORC recruits cdc6 and cdt1, followed by double-hexamer MCM replicative helicase loading, resulting in pre-RC formation (origin licensing). (iv) Telomeric origin fires.

Article Snippet: Antibodies used in ChIP assays include rabbit polyclonal antibodies ORC2 (Bethyl), MCM3 (Abcam), histone H3K9me3 (Diagenode) or IgG (Cell Signaling).

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Control, Hybridization, Labeling, Staining, Sequencing

Journal: Cell reports

Article Title: TRF2 Mediates Replication Initiation within Human Telomeres to Prevent Telomere Dysfunction

doi: 10.1016/j.celrep.2020.108379

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used in ChIP assays include rabbit polyclonal antibodies ORC2 (Bethyl), MCM3 (Abcam), histone H3K9me3 (Diagenode) or IgG (Cell Signaling).

Techniques: Plasmid Preparation, Virus, Recombinant, Transfection, Blocking Assay, Purification, Gel Extraction, DNA Purification, shRNA, Software