rabbit anti orc2  (Bethyl)

Journal: Science Advances

Article Title: FBXO42 facilitates Notch signaling activation and global chromatin relaxation by promoting K63-linked polyubiquitination of RBPJ

doi: 10.1126/sciadv.abq4831

Figure Lengend Snippet: ( A ) WT and FBXO42 KO cells were cotransfected with cSFB-RBPJ– and Myc-tagged constructs encoding epigenetically modified proteins. Then, cell lysates were incubated with S-protein beads and blotted with antibodies against FLAG- or MYC-epitope tags. ( B ) WT and FBXO42 KO cells were cotransfected with cSFB-RBPJ– and Myc-tagged constructs encoding SWI/SNF complex proteins. Then, the cells were harvested and analyzed as described in (A). ( C ) Heatmap showing the differential interaction between chromatin factors and RBPJ in WT and FBXO42 KO cells as identified by MS. ( D ) Enrichment analysis of the differentially interacting proteins of heterochromatin components is shown on the basis of GO annotation. ( E ) Immunofluorescence detection of HP1α foci in WT and FBXO42 KO cells. Scale bars, 10 μm. ( F ) HP1α foci number and percentage of HP1α foci area were calculated using ImageJ software. ( G ) WT and FBXO42 KO cells were digested with micrococcal nuclease (MNase) for 3 min, and chromatin relaxation was monitored by the release of nucleosomes. ( H ) Chromatin association of the SWI/SNF subunits SMARCA2, SMARCA4, and SMARCC2 in WT and FBXO42 KO cells was analyzed using Western blotting after chromatin isolation. ORC2 served as the loading control. ( I ) WT and FBXO42 KO cells were digested with deoxyribonuclease (DNase) for 3 min and followed with agarose gel electrophoresis analysis. ( J ) Chromatin from WT and FBXO42 KO cells was isolated, and DNase I was digested and used as substrate for accessibility assay. ( K and L ) The heatmap view for ATAC-seq signal intensity at TSSs in WT and FBXO42 KO JURKAT cells. ( M ) ATAC-seq peaks; H3K4m1, H3K4m3, and H3K27ac ChIP-seq peaks; and DNase-seq peaks downloaded from ENCODE database at MYC locus were analyzed. (A), (B), and (E) to (J), n = 3. Quantitative data are presented as means ± SEM. P values were calculated using two-tailed Student’s t tests. * P < 0.05 and ** P < 0.01.

Article Snippet: The following primary antibodies were used: rabbit anti-RBPJ [5313S, Cell Signaling Technology (CST), RRID:AB_2665555], mouse anti-FBXO42 (TA800283, OriGene, RRID:AB_2625356), THE hemagglutinin (HA) Tag (A01244, GenScript), THE c-Myc Tag (A00704, GenScript), ANTI-FLAG M2 antibody (B3111, Sigma-Aldrich, RRID:AB_2910145), rabbit anti-ubiquitin (AF0306, Beyotime), rabbit anti–β-actin (AC026, ABclonal, RRID:AB_2768234), rabbit anti-LSD1 (YM0422, ImmunoWay), rabbit anti-SMARCA4 (ET1611-85, HUABIO), rabbit anti-SMARCA2 (ER65406, HUABIO), rabbit anti-SMARCC2 (ER62787, HUABIO), and rabbit anti-origin recognition complex subunit 2 (ORC2) (A15697, ABclonal).

Techniques: Construct, Modification, Incubation, Immunofluorescence, Software, Western Blot, Isolation, Agarose Gel Electrophoresis, ChIP-sequencing, Two Tailed Test

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: Rabbit polyclonal anti-ORC2 , Bethyl , Cat# A302-735A: RRID:AB_10627808.

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

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