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ice cold lysis buffer b  (Nacalai)

 
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    Structured Review

    Nacalai ice cold lysis buffer b
    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

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    Related Articles

    Cell Culture:

    Article Title: 6PPD quinone induces cytokine production in fish cells via oxidative stress
    Article Snippet: N-(1,3-dimethylbutyl)-N -phenyl-p-phenylenediamine (6PPD) is widely used as an additive in tire manufacturing.. 6PPD quinone (6PPDQ), a derivative of 6PPD formed in the environment, is toxic to salmonid fish, particularly to coho salmon (Oncorhynchus kisutch).. The sensitivity to 6PPDQ varies among fish species at the cellular level.

    Modification:

    Article Title: ZIP13 marks muscle satellite cells and contributes to their quiescent and active phase balance.
    Article Snippet: .. Mononuclear cells were re-suspended in Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 2% Horse Serum (HS) (Sigma-Aldrich, MO, USA), 1% penicillin/streptomycin/ Amphotericin (PSA) (Antibiotic-Antimycotic Mixed Stock Solution (100x), 1000units/ml Penicillin, 1000 μg/ ml Streptomycin, 25 μg/ml Amphotericin B in 0.14% NaCl, 10nmol/L Citrate buffer; Stabilized) (nacalai tesque Fig. 6. ..

    Article Title: ZIP13 marks muscle satellite cells and contributes to their quiescent and active phase balance
    Article Snippet: .. Muscle slurry was prepared from the hindlimb muscles, thigh, and spine muscles of 3- 6-weeks-old male C57BL/6, Zip13-GFP -KI, and Zip13- KO mice, as described by Gromova A. et al. . Mononuclear cells were re-suspended in Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 2% Horse Serum (HS) (Sigma-Aldrich, MO, USA), 1% penicillin/streptomycin/Amphotericin (PSA) (Antibiotic-Antimycotic Mixed Stock Solution (100x), 1000units/ml Penicillin, 1000 μg/ml Streptomycin, 25 μg/ml Amphotericin B in 0.14% NaCl, 10nmol/L Citrate buffer; Stabilized) (nacalai tesque inc, Kyoto, Japan). .. Cells were sorted using a BD FACSMelody Cell sorter (BD Biosciences, NJ, USA).

    Lysis:

    Article Title: Regulation of brain-specific kinases 1 and 2 (BRSK1/2) by Ca 2+ /calmodulin.
    Article Snippet: Expression plasmids of His6-LKB1 (pME-His6-LKB1, 3 μg), HA-MO25 (pME-HA-MO25, 3 μg), and HA-STRAD (pME-HASTRAD, 3 μg) were transfected into COS-7 cells (10 cm dish, 6 plates) using polyethylenimine “MAX” (Polysciences, Inc., Warrington, PA, USA) according to the manufacturer’s protocol. .. After 48 h, the cells were lysed with ice-cold lysis buffer B (150 mM NaCl, 50 mM Tris–HCl [pH 7.5], 1 mM DTT, 10 mM imidazole, and 0.1% protease inhibitor cocktail [Nacalai Tesque; Kyoto, Japan]), followed by purification by Nisepharose chromatography according to the manufacturer’s protocol (Cytiva). .. Fractions eluted using a 300 mM imidazole-containing buffer B were dialyzed against 150 mM NaCl, 50 mM Tris–HCl (pH 7.5), and 1 mM DTT, followed by concentration using Amicon Ultra-0.5 mL (10 kDa MW cut, Merck Millipore Ltd, Tullagreen, IRL).

    Protease Inhibitor:

    Article Title: Regulation of brain-specific kinases 1 and 2 (BRSK1/2) by Ca 2+ /calmodulin.
    Article Snippet: Expression plasmids of His6-LKB1 (pME-His6-LKB1, 3 μg), HA-MO25 (pME-HA-MO25, 3 μg), and HA-STRAD (pME-HASTRAD, 3 μg) were transfected into COS-7 cells (10 cm dish, 6 plates) using polyethylenimine “MAX” (Polysciences, Inc., Warrington, PA, USA) according to the manufacturer’s protocol. .. After 48 h, the cells were lysed with ice-cold lysis buffer B (150 mM NaCl, 50 mM Tris–HCl [pH 7.5], 1 mM DTT, 10 mM imidazole, and 0.1% protease inhibitor cocktail [Nacalai Tesque; Kyoto, Japan]), followed by purification by Nisepharose chromatography according to the manufacturer’s protocol (Cytiva). .. Fractions eluted using a 300 mM imidazole-containing buffer B were dialyzed against 150 mM NaCl, 50 mM Tris–HCl (pH 7.5), and 1 mM DTT, followed by concentration using Amicon Ultra-0.5 mL (10 kDa MW cut, Merck Millipore Ltd, Tullagreen, IRL).

    Purification:

    Article Title: Regulation of brain-specific kinases 1 and 2 (BRSK1/2) by Ca 2+ /calmodulin.
    Article Snippet: Expression plasmids of His6-LKB1 (pME-His6-LKB1, 3 μg), HA-MO25 (pME-HA-MO25, 3 μg), and HA-STRAD (pME-HASTRAD, 3 μg) were transfected into COS-7 cells (10 cm dish, 6 plates) using polyethylenimine “MAX” (Polysciences, Inc., Warrington, PA, USA) according to the manufacturer’s protocol. .. After 48 h, the cells were lysed with ice-cold lysis buffer B (150 mM NaCl, 50 mM Tris–HCl [pH 7.5], 1 mM DTT, 10 mM imidazole, and 0.1% protease inhibitor cocktail [Nacalai Tesque; Kyoto, Japan]), followed by purification by Nisepharose chromatography according to the manufacturer’s protocol (Cytiva). .. Fractions eluted using a 300 mM imidazole-containing buffer B were dialyzed against 150 mM NaCl, 50 mM Tris–HCl (pH 7.5), and 1 mM DTT, followed by concentration using Amicon Ultra-0.5 mL (10 kDa MW cut, Merck Millipore Ltd, Tullagreen, IRL).

    Chromatography:

    Article Title: Regulation of brain-specific kinases 1 and 2 (BRSK1/2) by Ca 2+ /calmodulin.
    Article Snippet: Expression plasmids of His6-LKB1 (pME-His6-LKB1, 3 μg), HA-MO25 (pME-HA-MO25, 3 μg), and HA-STRAD (pME-HASTRAD, 3 μg) were transfected into COS-7 cells (10 cm dish, 6 plates) using polyethylenimine “MAX” (Polysciences, Inc., Warrington, PA, USA) according to the manufacturer’s protocol. .. After 48 h, the cells were lysed with ice-cold lysis buffer B (150 mM NaCl, 50 mM Tris–HCl [pH 7.5], 1 mM DTT, 10 mM imidazole, and 0.1% protease inhibitor cocktail [Nacalai Tesque; Kyoto, Japan]), followed by purification by Nisepharose chromatography according to the manufacturer’s protocol (Cytiva). .. Fractions eluted using a 300 mM imidazole-containing buffer B were dialyzed against 150 mM NaCl, 50 mM Tris–HCl (pH 7.5), and 1 mM DTT, followed by concentration using Amicon Ultra-0.5 mL (10 kDa MW cut, Merck Millipore Ltd, Tullagreen, IRL).

    other:

    Article Title: Establishment of a Novel Caco‐2‐Based Cell Culture System for Human Sapovirus Propagation
    Article Snippet: Protein bands were visualized using Chemi‐Lumi One L Solution A/B (Nacalai Tesque) and imaged with a ChemiDoc Touch system (Bio‐Rad).

    Article Title: Cell-intrinsic regulation of HBV RNAs by the nonsense-mediated mRNA decay pathway controls viral replication
    Article Snippet: Human sodium taurocholate co-transporting polypeptide (NTCP)-expressing HepG2 cells (NTCP/G2) were grown in William’s E medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% antibiotic-antimycotic solution (100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B; Nacalai Tesque), 2 mM L-glutamine (Nacalai Tesque), 50 μM hydrocortisone (Sigma-Aldrich), 5 μg/ml insulin (Sigma-Aldrich), 10 ng/ml EGF (Thermo Fisher Scientific), 5 μg/mL holo-transferrin (Wako), 5 ng/ml sodium selenite (Sigma-Aldrich) (primary hepatocyte maintain media: PMM), and 0.5 mg/ml G418 (Nacalai Tesque).

    Article Title: Cell-intrinsic regulation of HBV RNAs by the non-sense mediated mRNA decay pathway controls viral replication
    Article Snippet: Human sodium taurocholate co-transporting polypeptide (NTCP)-expressing HepG2 cells (NTCP/G2)31 were grown in William’s E medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% antibiotic-antimycotic solution (100 units/ mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B; Nacalai Tesque), 2 mM L-glutamine (Nacalai Tesque), 50 μM hydrocortisone (Sigma-Aldrich), 5 μg/ml insulin (Sigma-Aldrich), 10 ng/ml EGF (Thermo Fisher Scientific), 5 μg/mL holo-transferrin (Wako), 5 ng/ml sodium selenite (Sigma-Aldrich) (primary hepatocyte maintain media: PMM), and 0.5 mg/ml G418 (Nacalai Tesque).

    Muscles:

    Article Title: ZIP13 marks muscle satellite cells and contributes to their quiescent and active phase balance
    Article Snippet: .. Muscle slurry was prepared from the hindlimb muscles, thigh, and spine muscles of 3- 6-weeks-old male C57BL/6, Zip13-GFP -KI, and Zip13- KO mice, as described by Gromova A. et al. . Mononuclear cells were re-suspended in Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 2% Horse Serum (HS) (Sigma-Aldrich, MO, USA), 1% penicillin/streptomycin/Amphotericin (PSA) (Antibiotic-Antimycotic Mixed Stock Solution (100x), 1000units/ml Penicillin, 1000 μg/ml Streptomycin, 25 μg/ml Amphotericin B in 0.14% NaCl, 10nmol/L Citrate buffer; Stabilized) (nacalai tesque inc, Kyoto, Japan). .. Cells were sorted using a BD FACSMelody Cell sorter (BD Biosciences, NJ, USA).



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    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