Name: fmoc l arg pbf oh
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fmoc l arg pbf oh (Chem Impex International)

Chem Impex International fmoc l arg pbf oh
Fmoc L Arg Pbf Oh, supplied by Chem Impex International, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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fmoc l arg pbf oh - by Bioz Stars, 2026-02

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mus81 (Santa Cruz Biotechnology)

Santa Cruz Biotechnology mus81

Figure 3. SETX is essential for restarting R-loop-stalled forks via <t>MUS81-LIG4-ELL</t> 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.

Mus81, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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.

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: Antibodies used: Senataxin (rabbit polyclonal, Bethyl, A301-104A; 1:1000), Phospho-CHK1 (Ser345) (rabbit polyclonal, Cell Signalling, 2341S, 1:1000), CHK1 (mouse monoclonal, Santa Cruz Biotechnology, sc515369, 1:500), FLAG (mouse monoclonal, Sigma, F1804; 1:1000), TFIIH (rabbit polyclonal, Santa Cruz Biotechnology, sc-293; 1:2000), GAPDH (mouse monoclonal, Santa Cruz Biotechnology, sc-47724; 1:2000), MUS81 (mouse monoclonal, Santa Cruz Biotechnology, sc-53382; 1:500), GFP (rabbit polyclonal, Abcam, ab290; 1:2000), BRCA2 (mouse monoclonal, Sigma, OP95; 1:500), ZRANB3 (rabbit polyclonal, LindCare, 23111–1-AP; 1:1000), DDX17 (mouse monoclonal, Santa Cruz Biotechnology, sc-398168; 1:500), goat anti-rabbit IgG HRP (Sigma A0545; 1:10 000) and goat anti-mouse IgG HRP (Sigma A4416; 1:10 000).

Techniques: Inhibition, Labeling

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).

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).

Techniques: Degradation Assay, Transfection, Labeling, Western Blot

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).

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).

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

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.

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.

Techniques: Generated

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