rabbit polyclonal anti setx antibody  (Bethyl)

Journal: Nucleic Acids Research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: SETX forms a complex with DDX17 in human cells. ( A ) DDX17 isoforms (p72 and p82) co-immunoprecipitate with SETX from HEK293 cell extracts. Cells were treated either with 100 nM CPT or with vehicle (DMSO) for 1 h before harvest. Cell extracts were immunoprecipitated with rabbit polyclonal anti-SETX antibody. Rabbit IgG was incubated with extract of DMSO-treated cells as a control. Representative western blot of three independent experiments is shown. ( B ) Representative images (left panel) and quantification (right panel) of PLA foci between SETX and DDX17 in U2OS T-REx [RNaseH1-GFP] cells before and 24 h after induction of RNaseH1 expression with doxycycline (Dox). Medians of data sets from three independent experiments are plotted (n > 200). Data are presented as mean ± SD. Statistical analysis: Ordinary one-way ANOVA followed by Šídák's multiple comparisons, with a single pooled variance. Scale bar, 10 μ m.

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4°C.

Techniques: Immunoprecipitation, Incubation, Control, Western Blot, Expressing

Journal: Nucleic acids research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: Figure 1. SETX-depleted cells exhibit R-loop-mediated replication stress. ( A ) Western blot analysis to c hec k SETX expression levels and CHK1-S345 phosphorylation in U2OS cells transfected with two different siRNAs against SETX for 72 h. ( B ) Western blot analysis to confirm over-expression of RNaseH1-GFP in U2OS T-REx [RNaseH1-GFP] cells treated with do xy cy cline (Do x; 1 ng / ml) f or 24 h. ( C ) Schematic of DNA fiber labeling and quantification of replication tract lengths in mock (siLUC)- and SETX-depleted U2OS T-REx [RNaseH1-GFP] cells. 1 ng / ml Do x w as added 24 h before the labeling to induce RNase H1-GFP o v er-e xpression. Dimeth ylsulpho xide (DMSO) w as used as a v ehicle. T he v alues of CldU + IdU tract lengths measured in three independent experiments are plotted (n > 300). Red lines represent the median. ( D ) Quantification of replication fork stalling events on DNA fibers in ( C ). Replication tracts containing only the first label (CldU-only tracts) were designated as stalled replication forks while the replication tracts containing both labels (CldU + IdU) were scored as ongoing forks. Data represent mean ± SD ( n = 3). ( E ) Representative images and quantification of RNaseH1(D210N)-GFP foci in U2OS T-REx [RNaseH1(D210N)-GFP] cells transfected with indicated siRNAs. PCNA immunofluorescence staining was used to identify S-phase cells (PCNA + ). ( F ) R epresentativ e images and quantification of PLA foci between PCNA and elongating form of RNA polymerase II (RNAPIIS2P) in U2OS cells transfected with indicated siRNAs. EdU incorporation was performed to mark S-phase cells. Triptolide (1 μM) was added 2 h prior to PLA. (E, F) Medians of data sets from three independent experiments are plotted (n > 200). Data are presented as mean ± SD. Scale bar, 10 μm . Statistical analysis: Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used in ( C ). Ordinary one-way ANO V A f ollo w ed b y Šídák’s multiple comparisons, with a single pooled v ariance w as used in (D)–(F).

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4 ◦C.

Techniques: Western Blot, Expressing, Phospho-proteomics, Transfection, Over Expression, Labeling, Immunofluorescence, Staining

Journal: Nucleic acids research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: Figure 3. SETX is essential for restarting R-loop-stalled forks via MUS81-LIG4-ELL axis. ( A ) Effect of SETX depletion on replication fork recovery after induction of R-loop-mediated fork stalling in U2OS cells. Schematic of DNA fiber assays is shown on the top. The R-loop-inducing drug, camptothecin (CPT; 100 nM) was added to cells during CldU incorporation for the last 20 min. Dimethylsulphoxide (DMSO) was used as a vehicle. Cells were then washed and released into a fresh medium containing IdU. Replication tracts containing only CldU (red-only tracts) were designated as stalled replication f orks. R eplication tracts containing both CldU and IdU w ere scored as ongoing f orks (red / green tracts). T he percent age of st alled replication forks is plotted. Data represent mean ± SD ( n = 3). ( B ) Effect of SETX depletion on the rescue of CPT-induced fork stalling by PARP inhibition in U2OS cells, which requires the MUS81-LIG4-ELL axis. Schematic of DNA fiber assa y s and representativ e images of DNA replication tracts upon indicated conditions are shown on the left. CPT (100 nM) was present during IdU labeling. The PARP inhibitor (PARPi), olaparib (10 μM) was added 2 h before DNA fiber labeling and was also present during the labeling to boost replication restart. The values of the IdU / CldU tract length ratio obtained in three independent experiments are plotted ( n > 300). Red lines represent the median. Statistical analysis: Ordinary one-way ANO V A followed by Šídák’s multiple comparisons, with a single pooled variance was used in (A). Kruskal–Wallis test f ollo w ed b y Dunn’s multiple comparisons test w as used in (B). Scale bar, 10 μm.

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4 ◦C.

Techniques: Inhibition, Labeling

Journal: Nucleic acids research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: Figure 2. SETX depletion induces R-loop-dependent mitotic DNA synthesis and micronucleation. ( A ) Schematic representation of MiDAS assay, representative images and quantification of EdU incorporation events on metaphase chromosome spreads of U2OS T-REx [RNaseH1-GFP] cells transfected with indicated siRNAs. Cells were treated with 9 μM CDK1 inhibitor RO-3306 for 16 h, and then released into mitosis in the presence of 20 μM EdU and colcemid. Where indicated, o v er-e xpression of RNaseH1 was induced by the addition of doxycycline (Dox; 1 ng / ml) for 24 h. Dimeth ylsulpho xide (DMSO) was used as a vehicle. Data represent mean ± SD ( n = 3). At least 50 metaphase spreads were analyzed for the presence of EdU foci in each experiment. Scale bar, 10 μm . ( B ) Experimental w orkflo w with a representative image and quantification of micronuclei in U2OS T-REx [RNaseH1-GFP] cells transfected with indicated siRNAs. Cytochalasin B (2 μg / ml) was added 16 h prior to fixation to arrest cells before cytokinesis. Dox (1 ng / ml) was added to cells to induce RNaseH1-GFP over-expression. Data represent mean ± SD ( n = 4). At least 300 binucleated cells were scored in each experiment. Scale bar, 10 μm . Red arrow indicates micronucleus. ( C ) Experimental workflow with a representative image and quantification of 53BP1 nuclear bodies in U2OS T-REx [RNaseH1-GFP] cells transfected with indicated siRNAs. EdU incorporation was used to exclude S-phase cells from quantification. Dox (1 ng / ml) was added to cells to induce RNaseH1-GFP over-expression. Data represent mean ± SD ( n = 3). At least 300 EdU-negative cells were analyzed in each experiment. Scale bar, 10 μm . Statistical analysis: Ordinary one-way ANO V A followed by Šídák’s multiple comparisons, with a single pooled variance was used in (A)–(C).

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4 ◦C.

Techniques: DNA Synthesis, Transfection, Over Expression

Journal: Nucleic acids research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: Figure 4. SETX depletion induces MUS81-dependent nascent DNA degradation at R-loop-stalled forks. ( A ) Schematic of fork degradation assay. U2OS T-REx [RNaseH1-GFP] or U2OS cells were transfected with appropriate siRNA and after 72 h were subjected to DNA fiber labeling, followed by h y dro xyurea (HU; 4 mM) treatment for 5 h to induce replication fork st alling . Represent ative images of replication tracts on DNA fibers of mock (siLUC)- and SETX-depleted (siSETX-1) U2OS cells are also shown. Scale bar, 10 μm. ( B ) Quantification of HU-induced nascent DNA degradation in U2OS T-REx [RNaseH1-GFP] cells transfected with indicated siRNAs. Where indicated, do xy cy cline (Do x; 1 ng / ml) w as added 24 h prior to labeling to induce RNaseH1-GFP o v er-e xpression, which eliminates R-loops. (C–F) Quantification of HU-induced nascent DNA degradation in U2OS cells transfected with indicated siRNAs. Where indicated in ( C ), transcription inhibitors, DRB (100 μM) or triptolide (TRP, 1 μM), were added 2 h prior to labeling and were present throughout the labeling and HU treatment. In ( D ), SETX or BRCA2 were co-depleted with either ZRANB3 or HLTF to prevent fork reversal. Bottom panel shows western blot to confirm the depletion of indicated proteins. In ( E ), SETX was co-depleted with MUS81 to prevent fork cleavage. B ottom panel sho ws w estern blot to confirm the depletion of proteins. Where indicated in ( F ), MRE11 inhibitor, Mirin (50 μM) and the DNA2 inhibitor, C5 (25 μM) were added along with HU. In (B)–(F), the values of the IdU / CldU tract length ratio obtained in three independent experiments are plotted ( n > 300). Red lines represent the median. Statistical analysis: Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used in (B)–(F).

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4 ◦C.

Techniques: Degradation Assay, Transfection, Labeling, Western Blot

Journal: Nucleic acids research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: Figure 5. R eactiv ation of R-loop-stalled f orks via MUS81–LIG4–ELL axis depends on helicase activity of SETX. ( A ) Western blot analy sis of e xtracts of U2OS T-REx cells carrying siRNA-resistant wild-type (WT) or mutant (K1969R) SETX transgenes under the control of do xy cy cline-regulated CMV promoter. Cells were transfected with siLUC or siSETX-1 and cultured for 72 h. Where indicated, doxycycline (Dox, 10 ng / ml) was added 24 h before harvest to induce transgene expression. TFIIH served as a loading control. ( B ) Effect of expression of WT- or helicase-dead SETX transgene on replication fork progression in U2OS T-REx cells depleted of endogenous SETX. The values of CldU + IdU tract lengths measured in three independent experiments are plotted ( n > 300). Red lines represent the median. ( C ) Micronucleus frequency in U2OS T-REx cells expressing WT- or helicase-dead SETX transgene. Cells were transfected with siSETX-1 as in (A) to deplete endogenous SETX. Cytochalasin B was added 16 h prior to fixation to arrest cells before cytokinesis. Data represent mean ± SD ( n = 6). At least 300 binucleated cells were scored in each experiment. ( D ) Effect of PARP inhibition (PARPi) on CPT-induced fork stalling in U2OS T-REx cells expressing WT- or helicase-dead SETX transgene. The values of the IdU / CldU tract length ratio obtained in three independent experiments are plotted (n > 300). Red lines represent the median. ( E ) Effect of expression of WT- or helicase-dead SETX transgenes on HU-induced nascent DNA degradation in U2OS T-REx cells depleted of endogenous SETX. The values of the IdU / CldU tract length ratio obtained in three independent experiments are plotted (n > 300). Red lines represent the median. ( F ) R-loop levels in U2OS T-REx cells expressing the WT- or helicase-dead SETX transgene. S9.6 antibody was used to pull-down DNA fragments containing RNA:DNA hybrids and the enrichment of R-loop-prone loci (APOE, RPL13A and BTBD19) was measured by qPCR. SNRPN locus was used as a negative control for R-loop enrichment. Data represent mean ± SD ( n = 4). Statistical analysis: Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used in (B), (D) and (E). Ordinary one-w a y ANO V A f ollo w ed b y Šídák’s multiple comparisons, with a single pooled v ariance w as used in (C).

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4 ◦C.

Techniques: Activity Assay, Western Blot, Mutagenesis, Control, Transfection, Cell Culture, Expressing, Inhibition, Negative Control

Journal: Nucleic acids research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: Figure 6. Epistatic relationship between SETX and DDX17 in resolution of R-loop-mediated replication stress. ( A ) Western blot analysis to confirm depletion of both isoforms of DDX17, p72 and p82, in wild-type (WT) and SETX knockout U2OS cells, 72 h post siRNA transfection. ( B ) Effect of DDX17 depletion on replication fork progression in WT and SETX knockout U2OS cells. Schematic of DNA fiber assay is shown on the top. The values of CldU + IdU tract lengths measured in three independent experiments are plotted ( n > 300). Red lines represent the median. ( C ) The frequency of stalled replication forks in cells in (B). CldU replication tracts that are not followed by an IdU tract were considered as stalled forks. Data represent mean ± SD ( n = 4). ( D ) Effect of DDX17 depletion on nascent DNA degradation in WT and SETX knockout U2OS cells following HU (4 mM) treatment. Schematic of the fork degradation assay is shown on the top. The values of the IdU / CldU tract length ratio obtained in three independent experiments are plotted ( n > 300). Red lines represent the median. ( E ) Effect of DDX17 depletion of R-loop le v els in WT and SETX knockout U2OS cells. S9.6 antibody was used to pull-down DNA fragments containing RNA:DNA hybrids and the enrichment of R-loop-prone loci (APOE, RPL13A and BTBD19) was measured by qPCR. SNRPN locus was used as a negative control for R-loop enrichment. Data represent mean ± SD ( n = 3). ( F ) Effect of DDX17 depletion on micronucleus frequency in WT and SETX knockout cells. Cytochalasin B was added 16 h prior to fixation to arrest cells before cytokinesis. Data represent mean ± SD ( n = 3). At least 300 binucleated cells were scored in each experiment. Statistical analysis: Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used in (B) and (D). Ordinary one-way ANO V A followed by Šídák’s multiple comparisons, with a single pooled variance was used in (C) and (F).

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4 ◦C.

Techniques: Western Blot, Knock-Out, Transfection, Degradation Assay, Negative Control

Journal: Nucleic acids research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: Figure 8. Model for the role of SETX in restarting R-loop-stalled forks via the MUS81–LIG4–ELL pathway. To resume DNA replication, the stalled replication fork is cleaved by MUS81 endonuclease, which results in resolution of the torsional stress in the DNA generated by the transcription-replication encounter. SETX is proposed to act in a sequential manner with DDX17 helicase, possibly as a SETX-DDX17 complex (not shown), to mediate R-loop unwinding, a prerequisite for RNAPII transcription restart. In this process, DDX17 partially unwinds the RNA:DNA hybrid generating a 5 ′ -single-standed RNA tail where SETX is loaded to initiate processive unwinding of the RNA:DNA hybrid. DNA replication resumes f ollo wing f ork religation b y DNA ligase IV (LIG4) and ELL-mediated reactiv ation of transcription. SETX or DDX17 deficiency leads to accumulation of MUS81-clea v ed f orks, which are subjected to DNA2-mediated nucleolytic processing resulting in genomic inst abilit y. CMG, CDC45–MCM2-7–GINS helicase; Pol ε , DNA polymerase epsilon; Pol δ, DNA polymerase delta; RNAPII, RNA polymerase II.

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4 ◦C.

Techniques: Generated

Journal: Nucleic acids research

Article Title: Senataxin RNA/DNA helicase promotes replication restart at co-transcriptional R-loops to prevent MUS81-dependent fork degradation.

doi: 10.1093/nar/gkae673

Figure Lengend Snippet: Figure 7. SETX forms a complex with DDX17 in human cells. ( A ) DDX17 isoforms (p72 and p82) co-immunoprecipitate with SETX from HEK293 cell extracts. Cells were treated either with 100 nM CPT or with vehicle (DMSO) for 1 h before harvest. Cell extracts were immunoprecipitated with rabbit polyclonal anti-SETX antibody. Rabbit IgG was incubated with extract of DMSO-treated cells as a control. Representative western blot of three independent experiments is shown. ( B ) Representative images (left panel) and quantification (right panel) of PLA foci between SETX and DDX17 in U2OS T-REx [RNaseH1-GFP] cells before and 24 h after induction of RNaseH1 expression with doxycycline (Dox). Medians of data sets from three independent experiments are plotted (n > 200). Data are presented as mean ± SD. Statistical analysis: Ordinary one-way ANO V A followed by Šídák’s multiple comparisons, with a single pooled variance. Scale bar, 10 μm.

Article Snippet: Subsequently, 1200 μg of protein from the supernatant was incubated with 5 μg of rabbit polyclonal anti-SETX antibody (Bethyl, A301-104A), 250 units of benzonase nuclease (Sigma-Aldrich, E1014) and 5 mM MgCl 2 on a rotator at 4 ◦C.

Techniques: Immunoprecipitation, Incubation, Control, Western Blot, Expressing

Journal: Cell reports

Article Title: Dual Processing of R-Loops and Topoisomerase I Induces Transcription-Dependent DNA Double-Strand Breaks

doi: 10.1016/j.celrep.2019.08.041

Figure Lengend Snippet: (A and B) Quiescent cells were transfected with the indicated siRNAs and co-stained for γH2AX and p53BP1. To compare XPF-proficient (siCtrl) and XPF-deficient (siXPF) cells upon depletion of TDP1 (A) or SETX (B), γH2AX induction was normalized to 1 in both siCtrl and siXPF cells (means ± SEM; n = 3). **p < 0.01, ***p < 0.001 (two-tailed unpaired t test).

Article Snippet: SETX (Senataxin) rabbit polyclonal antibody (used for: WB) , Bethyl , Cat# A301-104A; RRID:AB_873128.

Techniques: Transfection, Staining, Two Tailed Test

Journal: Cell reports

Article Title: Dual Processing of R-Loops and Topoisomerase I Induces Transcription-Dependent DNA Double-Strand Breaks

doi: 10.1016/j.celrep.2019.08.041

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: SETX (Senataxin) rabbit polyclonal antibody (used for: WB) , Bethyl , Cat# A301-104A; RRID:AB_873128.

Techniques: Recombinant, Transfection, Viability Assay, Imaging, Luciferase, Control, Software, Fluorescence, Microscopy

Journal: Molecular Cell

Article Title: R-Loops Promote Antisense Transcription across the Mammalian Genome

doi: 10.1016/j.molcel.2019.10.002

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-SETX , Bethyl , Cat# A301-105A; RRID: AB_2186221.

Techniques: Virus, Recombinant, Mutagenesis, Sequencing, Plasmid Preparation, In Vitro, Biomarker Discovery, Dot Blot, Software