Review



wash buffer b  (Biosearch Technologies Inc)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 86
      Buy from Supplier

    Structured Review

    Biosearch Technologies Inc wash buffer b
    Wash Buffer B, supplied by Biosearch Technologies Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/a+buffer+wash/pmc12968391-111-9-12
    Average 86 stars, based on 1 article reviews
    wash buffer b - by Bioz Stars, 2026-09
    86/100 stars

    Images

    Related Articles

    Hybridization:

    Article Title: DDX5 orchestrates RNA homeostasis to ensure oocyte developmental competence.
    Article Snippet: .. After equilibration in Wash Buffer A (Biosearch Technologies) for 20 minutes at room temperature, hybridization was performed overnight at 37 °C in the dark using 500 nM oligo-dT30-Cy5 probes AR TI CL E IN P RE SS (synthesized by GenePharma) diluted in freshly prepared Hybridization Buffer (Biosearch Technologies). .. Following hybridization, oocytes were washed in Wash Buffer A for 30 minutes at 37 °C, stained with DAPI in fresh Wash Buffer A for 30 minutes at 37 °C, and then washed in Wash Buffer B (Biosearch Technologies) for 30 minutes at 37 °C.

    Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity
    Article Snippet: Cells were fixed with methanol/acetic acid at RT for 10 min, followed by washing with wash buffer A (Biosearch technologies, SMF-WA1-60). .. Hybridization was performed overnight in hybridization buffer (Biosearch technologies, SMF-HB1-10) with 10% formamide. .. After hybridization, cells were washed with wash buffer A for 30 min at 37°C and then blocked for 30 min using DAKO Protein Block Serum-Free Ready-to-Use (Agilent Dako, X0909).

    Article Title:
    Article Snippet: Fluorescein RNA Labeling Kit , Roche , Cat# 11685619910. .. smFISH Hybridization Buffer , Biosearch Technologies , Cat# SMF-HB1–10. .. Terminal Transferase , NEB , Cat# M0315S.

    Article Title: DDX5 orchestrates RNA homeostasis to ensure oocyte developmental competence
    Article Snippet: .. After equilibration in Wash Buffer A (Biosearch Technologies) for 20 min at room temperature, hybridization was performed overnight at 37 °C in the dark using 500 nM oligo-dT30-Cy5 probes (synthesized by GenePharma) diluted in freshly prepared Hybridization Buffer (Biosearch Technologies). .. Following hybridization, oocytes were washed in Wash Buffer A for 30 min at 37 °C, stained with DAPI in fresh Wash Buffer A for 30 min at 37 °C, and then washed in Wash Buffer B (Biosearch Technologies) for 30 min at 37 °C.

    Article Title: DDX5 orchestrates RNA homeostasis to ensure oocyte developmental competence.
    Article Snippet: After equilibration in Wash Buffer A (Biosearch Technologies) for 20 minutes at room temperature, hybridization was performed overnight at 37 °C in the dark using 500 nM oligo-dT30-Cy5 probes AR TI CL E IN P RE SS (synthesized by GenePharma) diluted in freshly prepared Hybridization Buffer (Biosearch Technologies). .. Following hybridization, oocytes were washed in Wash Buffer A for 30 minutes at 37 °C, stained with DAPI in fresh Wash Buffer A for 30 minutes at 37 °C, and then washed in Wash Buffer B (Biosearch Technologies) for 30 minutes at 37 °C. .. Imaging was performed using a Zeiss LSM880 confocal microscope.

    Synthesized:

    Article Title: DDX5 orchestrates RNA homeostasis to ensure oocyte developmental competence.
    Article Snippet: .. After equilibration in Wash Buffer A (Biosearch Technologies) for 20 minutes at room temperature, hybridization was performed overnight at 37 °C in the dark using 500 nM oligo-dT30-Cy5 probes AR TI CL E IN P RE SS (synthesized by GenePharma) diluted in freshly prepared Hybridization Buffer (Biosearch Technologies). .. Following hybridization, oocytes were washed in Wash Buffer A for 30 minutes at 37 °C, stained with DAPI in fresh Wash Buffer A for 30 minutes at 37 °C, and then washed in Wash Buffer B (Biosearch Technologies) for 30 minutes at 37 °C.

    Article Title: DDX5 orchestrates RNA homeostasis to ensure oocyte developmental competence
    Article Snippet: .. After equilibration in Wash Buffer A (Biosearch Technologies) for 20 min at room temperature, hybridization was performed overnight at 37 °C in the dark using 500 nM oligo-dT30-Cy5 probes (synthesized by GenePharma) diluted in freshly prepared Hybridization Buffer (Biosearch Technologies). .. Following hybridization, oocytes were washed in Wash Buffer A for 30 min at 37 °C, stained with DAPI in fresh Wash Buffer A for 30 min at 37 °C, and then washed in Wash Buffer B (Biosearch Technologies) for 30 min at 37 °C.

    other:

    Article Title: FUS is an N 1- and N 6 -methyladenosine-binding protein
    Article Snippet: Hybridization and washing buffers were obtained from Biosearch Technologies and used following the manufacturer’s protocol.

    Incubation:

    Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity
    Article Snippet: The cells were then incubated with Fluorescein Avidin DCS (5 μg/ml, Vector Laboratories, A-2011) for 30 min at RT. .. Slides were washed twice for 3 min each with wash buffer B (Biosearch technologies, SMF-WB1-20), followed by incubation with Biotinylated Anti-Avidin (5 μg/ml, Vector Laboratories, BA-0300) for 30 min at RT. ..

    Staining:

    Article Title: DDX5 orchestrates RNA homeostasis to ensure oocyte developmental competence.
    Article Snippet: After equilibration in Wash Buffer A (Biosearch Technologies) for 20 minutes at room temperature, hybridization was performed overnight at 37 °C in the dark using 500 nM oligo-dT30-Cy5 probes AR TI CL E IN P RE SS (synthesized by GenePharma) diluted in freshly prepared Hybridization Buffer (Biosearch Technologies). .. Following hybridization, oocytes were washed in Wash Buffer A for 30 minutes at 37 °C, stained with DAPI in fresh Wash Buffer A for 30 minutes at 37 °C, and then washed in Wash Buffer B (Biosearch Technologies) for 30 minutes at 37 °C. .. Imaging was performed using a Zeiss LSM880 confocal microscope.



    Similar Products

    rt buffer b  (10X Genomics)
    86
    10X Genomics rt buffer b
    Rt Buffer B, supplied by 10X Genomics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/pm42260119-394-14-17
    Average 86 stars, based on 1 article reviews
    rt buffer b - by Bioz Stars, 2026-09
    86/100 stars
    		  Buy from Supplier
    buffer b  (Fisher Scientific)
    86
    Fisher Scientific buffer b
    Buffer B, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/buffer+lysis/bio_rxiv__64898__2026__05__13__724352-122-9-16
    Average 86 stars, based on 1 article reviews
    buffer b - by Bioz Stars, 2026-09
    86/100 stars
    		  Buy from Supplier
    tris wash buffer b  (Zytomed Inc)
    86
    Zytomed Inc tris wash buffer b
    Tris Wash Buffer B, supplied by Zytomed Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/buffer+tris+wash/pm42122993-104-10-15
    Average 86 stars, based on 1 article reviews
    tris wash buffer b - by Bioz Stars, 2026-09
    86/100 stars
    		  Buy from Supplier
    ramos b cells  (Thermo Fisher)
    99
    Thermo Fisher ramos b cells
    Ramos B Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/RIPA+buffer/pm41998187-308-6-56
    Average 99 stars, based on 1 article reviews
    ramos b cells - by Bioz Stars, 2026-09
    99/100 stars
    		  Buy from Supplier
    buffer b trf2 rap1  (Bio-Rad)
    96
    Bio-Rad buffer b trf2 rap1
    <t>TRF2–RAP1</t> <t>interaction</t> enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.
    Buffer B Trf2 Rap1, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/Micro+Bio-Spin+6/pmc13036488-88-19-16
    Average 96 stars, based on 1 article reviews
    buffer b trf2 rap1 - by Bioz Stars, 2026-09
    96/100 stars
    		  Buy from Supplier
    wash buffer b  (Biosearch Technologies Inc)
    86
    Biosearch Technologies Inc wash buffer b
    <t>TRF2–RAP1</t> <t>interaction</t> enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.
    Wash Buffer B, supplied by Biosearch Technologies Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/a+buffer+wash/pmc12968391-111-9-12
    Average 86 stars, based on 1 article reviews
    wash buffer b - by Bioz Stars, 2026-09
    86/100 stars
    		  Buy from Supplier
    ice cold lysis buffer b  (Nacalai)
    86
    Nacalai ice cold lysis buffer b
    <t>TRF2–RAP1</t> <t>interaction</t> enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.
    Ice Cold Lysis Buffer B, supplied by Nacalai, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/buffer+ripa/pm41905287-73-8-31
    Average 86 stars, based on 1 article reviews
    ice cold lysis buffer b - by Bioz Stars, 2026-09
    86/100 stars
    		  Buy from Supplier
    b v co  (Miltenyi Biotec)
    95
    Miltenyi Biotec b v co
    <t>TRF2–RAP1</t> <t>interaction</t> enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.
    B V Co, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/buffer+b/MACSima+Running+Buffer/pmc12796729-42-6-4
    Average 95 stars, based on 1 article reviews
    b v co - by Bioz Stars, 2026-09
    95/100 stars
    		  Buy from Supplier

    Image Search Results


    TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

    doi: 10.1093/nar/gkag272

    Figure Lengend Snippet: TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.

    Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

    Techniques: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Purification, Mobility Shift

    TRF2–RAP1 promotes BLM-mediated unwinding of telomere R-loops. ( A ) (Top) Schematic of the oligo-based telomere R-loop unwinding assay. Telomere R-loop substrates were generated by hybridizing 32 P-labeled TERRA and two telomere DNA fragments (TDR2 and TDR3). TRF2 and/or RAP1 were pre-incubated with the R-loops and then BLM was added to the reaction, and the complex was resolved by 10% native polyacrylamide gel electrophoresis to monitor for R-loop unwinding. Displacement of the invading radiolabeled TERRA from R-loops indicates that R-loop unwinding. (Bottom) The TRF2–RAP1 complex promotes BLM-mediated unwinding of telomere R-loops. The effects of TRF2 alone (40, 80 nM) or in combination with RAP1 (20, 40, 80 nM) on the ability of BLM (20 nM) to unwind telomere R-loops were examined. 32 P-labeled TERRA and R-loops were resolved by native-PAGE and shown in lanes 1 and 2. ( B ) Quantification of BLM-mediated R-loop unwinding reactions in panel (A). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .9485); **** P < .0001. ( C ) The TRF2 basic domain is required for efficient unwinding of telomere R-loops. The effect of WT TRF2, TRF2 ΔB , TRF2 ΔB,L288R , and RAP1 to enhance BLM-mediated telomere R-loop unwinding was examined. The sizes of 32 P-labeled TERRA and R-loops were resolved by native-PAGE, as shown in lanes 1 and 2. ( D ) Quantification of BLM-mediated R-loop unwinding reactions in panel (C). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. **** P < .0001. ( E ) The TRF2–BLM interaction enhances telomere R-loop unwinding. The effect of TRF2–RAP1 on the ability of WT and mutant BLM (3A or P690L) to unwind telomere R-loops was tested as in Fig. . 32 P-labeled TERRA and R-loops were loaded as size markers (lanes 1 and 2) and resolved by native-PAGE. ( F ) Quantification of the percentages of unwound R-loops in panel (E) as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .98); **** P < .0001.

    Journal: Nucleic Acids Research

    Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

    doi: 10.1093/nar/gkag272

    Figure Lengend Snippet: TRF2–RAP1 promotes BLM-mediated unwinding of telomere R-loops. ( A ) (Top) Schematic of the oligo-based telomere R-loop unwinding assay. Telomere R-loop substrates were generated by hybridizing 32 P-labeled TERRA and two telomere DNA fragments (TDR2 and TDR3). TRF2 and/or RAP1 were pre-incubated with the R-loops and then BLM was added to the reaction, and the complex was resolved by 10% native polyacrylamide gel electrophoresis to monitor for R-loop unwinding. Displacement of the invading radiolabeled TERRA from R-loops indicates that R-loop unwinding. (Bottom) The TRF2–RAP1 complex promotes BLM-mediated unwinding of telomere R-loops. The effects of TRF2 alone (40, 80 nM) or in combination with RAP1 (20, 40, 80 nM) on the ability of BLM (20 nM) to unwind telomere R-loops were examined. 32 P-labeled TERRA and R-loops were resolved by native-PAGE and shown in lanes 1 and 2. ( B ) Quantification of BLM-mediated R-loop unwinding reactions in panel (A). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .9485); **** P < .0001. ( C ) The TRF2 basic domain is required for efficient unwinding of telomere R-loops. The effect of WT TRF2, TRF2 ΔB , TRF2 ΔB,L288R , and RAP1 to enhance BLM-mediated telomere R-loop unwinding was examined. The sizes of 32 P-labeled TERRA and R-loops were resolved by native-PAGE, as shown in lanes 1 and 2. ( D ) Quantification of BLM-mediated R-loop unwinding reactions in panel (C). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. **** P < .0001. ( E ) The TRF2–BLM interaction enhances telomere R-loop unwinding. The effect of TRF2–RAP1 on the ability of WT and mutant BLM (3A or P690L) to unwind telomere R-loops was tested as in Fig. . 32 P-labeled TERRA and R-loops were loaded as size markers (lanes 1 and 2) and resolved by native-PAGE. ( F ) Quantification of the percentages of unwound R-loops in panel (E) as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .98); **** P < .0001.

    Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

    Techniques: Generated, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Clear Native PAGE, Mutagenesis

    BLM preferentially releases TERRA over ssDNA from telomere D/R-loops. ( A ) Schematic of the assay used to measure how TRF2–RAP1 promotes BLM-mediated unwinding of RAD51/ssDNA and RAD51AP1/TERRA-generated telomeric D/R-loops. Telomere D/R-loops were generated by incubating RAD51 with IRDye-700-labeled telomere ssDNA (red), RAD51AP1 with IRDye-800-labeled TERRA (green), and telomere plasmids together as described in Fig. . Native plasmid-sized telomere D/R-loops were obtained after deproteinization and column purification. BLM with or without TRF2–RAP1 was then incubated with these D/R-loops, and ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. ( B ) BLM preferentially releases TERRA over ssDNA from telomere D/R-loop. BLM (20, 40, 80 nM) was tested for its ability to unwind telomere D/R-loops or TRF2–RAP1-bound D/R-loops. ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. The unwinding of telomere D/R-loops by BLM was enhanced by TRF2–RAP1. ( C ) Quantification of the amount of D- and R-loops relative to the negative control (no proteins, lane 1). Data were plotted as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. * P = .02282; ** P = .001278; *** P = .0007284; **** P < .0001. ( D ) The effects of TRF2–RAP1 on WT BLM, the helicase-dead BLM K695R or BLM mutants on D/R-loop unwinding were tested as in panel (B). In contrast to WT BLM, TRF2–RAP1 cannot enhance BLM ’s ability to unwind telomere D/R-loops. D/R-loop unwinding was analyzed by 1% agarose gels. ( E ) Quantification of the relative amounts of D-loops or R-loops to the control without proteins (lane 1) is shown as mean ± SD from three independent experiments. ANOVA test was used to evaluate statistical differences. ns: non-significant ( P = .15; .4147; .8026); ** P = .001193; *** P = .000158.

    Journal: Nucleic Acids Research

    Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

    doi: 10.1093/nar/gkag272

    Figure Lengend Snippet: BLM preferentially releases TERRA over ssDNA from telomere D/R-loops. ( A ) Schematic of the assay used to measure how TRF2–RAP1 promotes BLM-mediated unwinding of RAD51/ssDNA and RAD51AP1/TERRA-generated telomeric D/R-loops. Telomere D/R-loops were generated by incubating RAD51 with IRDye-700-labeled telomere ssDNA (red), RAD51AP1 with IRDye-800-labeled TERRA (green), and telomere plasmids together as described in Fig. . Native plasmid-sized telomere D/R-loops were obtained after deproteinization and column purification. BLM with or without TRF2–RAP1 was then incubated with these D/R-loops, and ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. ( B ) BLM preferentially releases TERRA over ssDNA from telomere D/R-loop. BLM (20, 40, 80 nM) was tested for its ability to unwind telomere D/R-loops or TRF2–RAP1-bound D/R-loops. ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. The unwinding of telomere D/R-loops by BLM was enhanced by TRF2–RAP1. ( C ) Quantification of the amount of D- and R-loops relative to the negative control (no proteins, lane 1). Data were plotted as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. * P = .02282; ** P = .001278; *** P = .0007284; **** P < .0001. ( D ) The effects of TRF2–RAP1 on WT BLM, the helicase-dead BLM K695R or BLM mutants on D/R-loop unwinding were tested as in panel (B). In contrast to WT BLM, TRF2–RAP1 cannot enhance BLM ’s ability to unwind telomere D/R-loops. D/R-loop unwinding was analyzed by 1% agarose gels. ( E ) Quantification of the relative amounts of D-loops or R-loops to the control without proteins (lane 1) is shown as mean ± SD from three independent experiments. ANOVA test was used to evaluate statistical differences. ns: non-significant ( P = .15; .4147; .8026); ** P = .001193; *** P = .000158.

    Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

    Techniques: Generated, Labeling, Plasmid Preparation, Purification, Incubation, Negative Control, Control

    TRF2–RAP1–BLM is required to resolve telomere R-loops in U2OS cells. ( A ) U2OS cells expressing TRF2 ΔB, L288R were treated with shControl, shBLM, or shTRF2. Immunofluorescence-FISH analysis of cells containing UTs (PNA telomere probe, red) co-localized with R-loops (S9.6 antibody, green) and DAPI-stained nuclei (blue). White arrow: co-localization of R-loops on UTs. U2OS cells expressing shBLM-resistant WT BLM cDNA and indicated BLM mutants were treated with shBLM, shTRF2, and TRF2 ΔB, L288R . IF-FISH analysis was performed to detect UT/R-loop co-localization. White arrow: co-localization of R-loops on UTs. ( C ) Quantification of data from Fig. and , showing the number of UT/R-loop colocalizations per U2OS cell. Data from three independent experiments is shown as mean ± SEM from minimum 200 nuclei per experiment. Statistical evaluation was performed by one-way ANOVA test. ns: non-significant ( P > .9999); ** P = .0032; .0035; .0062; .0092; .0052; **** P < .0001. ( D ) Model showing that TRF2–RAP1 inhibits telomere HDR by promoting BLM-mediated telomere R-loop removal. RAD51AP1 and TERRA-dependent R-loops promote RAD51-mediated telomere D-loop formation. The TRF2–RAP1 complex and TRF2–BLM interaction are required to promote BLM helicase-mediated unwinding of telomere R-loops and then D-loops. The RAP1–TRF2–BLM complex represses HDR on telomeres by removing R-loops to inhibit telomere D-loop formation.

    Journal: Nucleic Acids Research

    Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

    doi: 10.1093/nar/gkag272

    Figure Lengend Snippet: TRF2–RAP1–BLM is required to resolve telomere R-loops in U2OS cells. ( A ) U2OS cells expressing TRF2 ΔB, L288R were treated with shControl, shBLM, or shTRF2. Immunofluorescence-FISH analysis of cells containing UTs (PNA telomere probe, red) co-localized with R-loops (S9.6 antibody, green) and DAPI-stained nuclei (blue). White arrow: co-localization of R-loops on UTs. U2OS cells expressing shBLM-resistant WT BLM cDNA and indicated BLM mutants were treated with shBLM, shTRF2, and TRF2 ΔB, L288R . IF-FISH analysis was performed to detect UT/R-loop co-localization. White arrow: co-localization of R-loops on UTs. ( C ) Quantification of data from Fig. and , showing the number of UT/R-loop colocalizations per U2OS cell. Data from three independent experiments is shown as mean ± SEM from minimum 200 nuclei per experiment. Statistical evaluation was performed by one-way ANOVA test. ns: non-significant ( P > .9999); ** P = .0032; .0035; .0062; .0092; .0052; **** P < .0001. ( D ) Model showing that TRF2–RAP1 inhibits telomere HDR by promoting BLM-mediated telomere R-loop removal. RAD51AP1 and TERRA-dependent R-loops promote RAD51-mediated telomere D-loop formation. The TRF2–RAP1 complex and TRF2–BLM interaction are required to promote BLM helicase-mediated unwinding of telomere R-loops and then D-loops. The RAP1–TRF2–BLM complex represses HDR on telomeres by removing R-loops to inhibit telomere D-loop formation.

    Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

    Techniques: Expressing, Immunofluorescence, Staining