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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: Therapeutic potential of photoimmunotherapy in solid tumors expressing CD98 heavy chain.
    Article Snippet: .. Cultured cells were lysed in Cell Lysis Buffer (Nacalai Tesque, Kyoto, Japan), and lysates were centrifuged to remove debris. .. Samples were diluted in Sample Buffer Solution with Reducing Reagent for SDS-PAGE (Nacalai Tesque)

    Lysis:

    Article Title: Therapeutic potential of photoimmunotherapy in solid tumors expressing CD98 heavy chain.
    Article Snippet: .. Cultured cells were lysed in Cell Lysis Buffer (Nacalai Tesque, Kyoto, Japan), and lysates were centrifuged to remove debris. .. Samples were diluted in Sample Buffer Solution with Reducing Reagent for SDS-PAGE (Nacalai Tesque)

    Article Title: TMPRSS2-induced Golgi disruption restricts the incorporation of virus envelope glycoproteins into virions.
    Article Snippet: .. After 48 h of culture, culture supernatants were filtered through a 0.45-μm filter, and viruses were collected by centrifugation on 20% of sucrose (Sigma) in PBS at 30,000 rpm for 90 min at 4 °C using an SW41 rotor (Beckman Coulter, Brea, CA, USA) and lysed using RIPA lysis buffer (16488-34, Nacalai Tesque, Kyoto, Japan). .. Cells were lysed in RIPA buffer containing a complete proteinase inhibitor (05892953001; Roche, Basel, Switzerland).

    Article Title: AKIP1 is an inner scaffold component required for centriole integrity
    Article Snippet: .. HEK293T cells were collected 24 h after transfection and lysed on ice in lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.5% Triton X-100, 1 mM DTT, and 1:500 protease inhibitor cocktail (Nacalai Tesque, 25955-11)). .. The lysates were removed after centrifugation for 10 min. For IP of FLAG-tagged proteins, whole-cell lysates were incubated with M2 agarose gel conjugated with a FLAG antibody (Merck Millipore, A2220) for 2 h or overnight at 4°C.

    Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism
    Article Snippet: .. The collected cells were lysed with lysis buffer (20 mM HEPES-NaOH [pH 7.5] [Nacalai Tesque], 2.5 mM MgCl 2 , 150 mM NaCl, and 1% [v/v] Triton X-100) containing 1× cOmplete EDTA-free protease inhibitor cocktail, 1× PhosSTOP phosphatase inhibitor cocktail (Roche Diagnostics), 1 mM DTT, and 100 μg/ml CHX. ..

    Protease Inhibitor:

    Article Title: Dapagliflozin confers protection against ferroptosis in cardiomyocyte via the inhibition of interferon-gamma pathway.
    Article Snippet: The absorbance was recorded at 530 nm using Thermo ScientificTM MultiskanTM FC Microplate Photometer (Thermo Fisher Scientific, Massachusetts, USA). .. Treated cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). .. Cell lysates were incubated in a working reagent, consisting of glutathione reduction enzyme, nicotinamide adenine dinucleotide phosphate (NADPH), and 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma-Aldrich, St. Louis, USA).

    Article Title: AKIP1 is an inner scaffold component required for centriole integrity
    Article Snippet: .. HEK293T cells were collected 24 h after transfection and lysed on ice in lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.5% Triton X-100, 1 mM DTT, and 1:500 protease inhibitor cocktail (Nacalai Tesque, 25955-11)). .. The lysates were removed after centrifugation for 10 min. For IP of FLAG-tagged proteins, whole-cell lysates were incubated with M2 agarose gel conjugated with a FLAG antibody (Merck Millipore, A2220) for 2 h or overnight at 4°C.

    Article Title: Protective Effects of the Phytocannabinoid Cannabidiol on Disuse-Induced Muscle Atrophy through Modulation of Proteolysis and Mitochondrial Regulation.
    Article Snippet: .. Western Blotting Total protein was extracted with RIPA buffer containing 10 mM Tris–HCl (pH 7.4), 1% Nonidet P-40, 150 mM NaCl, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail (Nacalai Tesque). ..

    Article Title: Autoinhibition of the mechanosensitive lipid scramblase TMEM63B by its C-terminal tail.
    Article Snippet: When necessary, cells expressing the introduced constructs were sorted using a CytoFLEX SRT (Beckman Coulter). .. Cells were lysed in RIPA buffer (50 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate) containing a protease inhibitor cocktail (Nacalai Tesque). .. Protein concentrations were determined using a BCA assay (Nacalai Tesque).

    Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism
    Article Snippet: .. The collected cells were lysed with lysis buffer (20 mM HEPES-NaOH [pH 7.5] [Nacalai Tesque], 2.5 mM MgCl 2 , 150 mM NaCl, and 1% [v/v] Triton X-100) containing 1× cOmplete EDTA-free protease inhibitor cocktail, 1× PhosSTOP phosphatase inhibitor cocktail (Roche Diagnostics), 1 mM DTT, and 100 μg/ml CHX. ..

    Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism
    Article Snippet: .. The cells were lysed with Radio-Immunoprecipitation Assay (RIPA) buffer (10 mM Tris–HCl [pH 7.5] [Nacalai Tesque], 1 mM EDTA [Nacalai Tesque], 1% [v/v] Triton X-100 [Nacalai Tesque], 0.1% [w/v] sodium deoxycholate [Nacalai Tesque], 0.1% [w/v] SDS [Nacalai Tesque], and 140 mM NaCl [Nacalai Tesque]) containing 1× cOmplete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) on ice for 30 min. ..

    Centrifugation:

    Article Title: TMPRSS2-induced Golgi disruption restricts the incorporation of virus envelope glycoproteins into virions.
    Article Snippet: .. After 48 h of culture, culture supernatants were filtered through a 0.45-μm filter, and viruses were collected by centrifugation on 20% of sucrose (Sigma) in PBS at 30,000 rpm for 90 min at 4 °C using an SW41 rotor (Beckman Coulter, Brea, CA, USA) and lysed using RIPA lysis buffer (16488-34, Nacalai Tesque, Kyoto, Japan). .. Cells were lysed in RIPA buffer containing a complete proteinase inhibitor (05892953001; Roche, Basel, Switzerland).

    Transfection:

    Article Title: AKIP1 is an inner scaffold component required for centriole integrity
    Article Snippet: .. HEK293T cells were collected 24 h after transfection and lysed on ice in lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.5% Triton X-100, 1 mM DTT, and 1:500 protease inhibitor cocktail (Nacalai Tesque, 25955-11)). .. The lysates were removed after centrifugation for 10 min. For IP of FLAG-tagged proteins, whole-cell lysates were incubated with M2 agarose gel conjugated with a FLAG antibody (Merck Millipore, A2220) for 2 h or overnight at 4°C.

    Western Blot:

    Article Title: Protective Effects of the Phytocannabinoid Cannabidiol on Disuse-Induced Muscle Atrophy through Modulation of Proteolysis and Mitochondrial Regulation.
    Article Snippet: .. Western Blotting Total protein was extracted with RIPA buffer containing 10 mM Tris–HCl (pH 7.4), 1% Nonidet P-40, 150 mM NaCl, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail (Nacalai Tesque). ..

    Radio Immunoprecipitation:

    Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism
    Article Snippet: .. The cells were lysed with Radio-Immunoprecipitation Assay (RIPA) buffer (10 mM Tris–HCl [pH 7.5] [Nacalai Tesque], 1 mM EDTA [Nacalai Tesque], 1% [v/v] Triton X-100 [Nacalai Tesque], 0.1% [w/v] sodium deoxycholate [Nacalai Tesque], 0.1% [w/v] SDS [Nacalai Tesque], and 140 mM NaCl [Nacalai Tesque]) containing 1× cOmplete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) on ice for 30 min. ..



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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