hinfi (New England Biolabs)

New England Biolabs is a verified supplier

New England Biolabs manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

New England Biolabs hinfi

TbUMSBP2 knockdown altered the amount of single stranded telomeric <t>DNA.</t> DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with <t>HinfI</t> and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

Hinfi, supplied by New England Biolabs, 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/result/hinfi/product/New England Biolabs
Average 96 stars, based on 1 article reviews
Price from $9.99 to $1999.99

hinfi - by Bioz Stars, 2022-05

96/100 stars

Images

1) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky597

TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

Techniques Used: Hybridization

TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

2) Product Images from "WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening ▿-Mediated Telomere Shortening ▿ †"

Article Title: WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening ▿-Mediated Telomere Shortening ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01364-07

Telomeric circles are present in telomerase-positive WS fibroblasts in the absence of TRF2 ΔB . DNA isolated from normal (A) and WS (C) fibroblasts transduced with lentiviruses expressing the indicated proteins was digested with HinfI and RsaI, separated by size and shape, blotted, and probed with a telomeric (CCCTAA) repeat probe. Arrows indicate arcs of telomeric DNA circles. Circularized λ × HindIII DNA fragments were used as molecular size markers (the 23- and 4.4-kb fragments have one cos end and do not circularize). Samples shown in panels A and C were run and processed in parallel under the same hybridization and washing conditions. (B) DNA isolated from ALT fibroblasts was separated by 2DGE and probed with a telomeric (CCCTAA) 4 probe. The data shown are representative of at least three independent experiments. The approximate level of telomeric circles (expressed as a percentage of the total telomeric DNA) present in each sample was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

Figure Legend Snippet: Telomeric circles are present in telomerase-positive WS fibroblasts in the absence of TRF2 ΔB . DNA isolated from normal (A) and WS (C) fibroblasts transduced with lentiviruses expressing the indicated proteins was digested with HinfI and RsaI, separated by size and shape, blotted, and probed with a telomeric (CCCTAA) repeat probe. Arrows indicate arcs of telomeric DNA circles. Circularized λ × HindIII DNA fragments were used as molecular size markers (the 23- and 4.4-kb fragments have one cos end and do not circularize). Samples shown in panels A and C were run and processed in parallel under the same hybridization and washing conditions. (B) DNA isolated from ALT fibroblasts was separated by 2DGE and probed with a telomeric (CCCTAA) 4 probe. The data shown are representative of at least three independent experiments. The approximate level of telomeric circles (expressed as a percentage of the total telomeric DNA) present in each sample was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

Techniques Used: Isolation, Transduction, Expressing, Hybridization

TRF2 ΔB -induced cell senescence, TIFs, and telomere shortening are reconstituted in WS fibroblasts genetically complemented with wild-type WRN but not enzymatically deficient WRN variants. (A) Expression of wild-type and mutant forms of WRN in WS cells. WS cells were infected with lentiviruses for the expression wild-type, helicase-deficient, exonuclease-deficient, and helicase- and exonuclease-deficient forms of WRN and cultured for 2 weeks. The parental and genetically complemented cells lines were then transduced with a control virus or a virus for the expression of Flag-TRF2 ΔB or Flag-TRF2. Analysis of protein expression was performed by preparation of nuclear extracts, followed by Western blotting with anti-WRN (top panel), antitubulin (middle panel), and anti-Flag (bottom panel) antibodies. (B) Detection of SA-βgal activity. Telomerase-positive WS fibroblasts transduced with the indicated lentiviruses were cultured for 8 days, fixed, and stained for SA-βgal. Five hundred cells of each line were analyzed in duplicate plates. Each bar represents the mean ± the standard deviation of three independent experiments ( n = 3) carried out in duplicate. WT, wild type. (C) Detection of 53BP1 and TRF1 in WS fibroblasts consecutively transduced with lentiviruses expressing the indicated proteins with antibodies against 53BP1 (green) and TRF1 (red) 1 day after the second transduction. For the quantitation of 53BP1 foci and 53BP1 and TRF1 colocalization, see Fig. S1 in the supplemental material. (D) The parental and genetically complemented cell lines were transduced with control lentivirus (lanes 1 and 4) and lentiviruses for the expression of Flag-TRF2 ΔB (lanes 2 and 5) or Flag-TRF2 (lanes 3 and 6). Cells were harvested 8 days after lentivirus transduction, and genomic DNA was isolated and digested with HinfI and RsaI. Equal amounts (2 μg) of digested genomic DNA were separated by electrophoresis on a 0.8% agarose gel, followed by Southern blot analysis with a radiolabeled (TTAGGG) 3 probe. Southern blot analyses were performed on three independent samples of WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

Figure Legend Snippet: TRF2 ΔB -induced cell senescence, TIFs, and telomere shortening are reconstituted in WS fibroblasts genetically complemented with wild-type WRN but not enzymatically deficient WRN variants. (A) Expression of wild-type and mutant forms of WRN in WS cells. WS cells were infected with lentiviruses for the expression wild-type, helicase-deficient, exonuclease-deficient, and helicase- and exonuclease-deficient forms of WRN and cultured for 2 weeks. The parental and genetically complemented cells lines were then transduced with a control virus or a virus for the expression of Flag-TRF2 ΔB or Flag-TRF2. Analysis of protein expression was performed by preparation of nuclear extracts, followed by Western blotting with anti-WRN (top panel), antitubulin (middle panel), and anti-Flag (bottom panel) antibodies. (B) Detection of SA-βgal activity. Telomerase-positive WS fibroblasts transduced with the indicated lentiviruses were cultured for 8 days, fixed, and stained for SA-βgal. Five hundred cells of each line were analyzed in duplicate plates. Each bar represents the mean ± the standard deviation of three independent experiments ( n = 3) carried out in duplicate. WT, wild type. (C) Detection of 53BP1 and TRF1 in WS fibroblasts consecutively transduced with lentiviruses expressing the indicated proteins with antibodies against 53BP1 (green) and TRF1 (red) 1 day after the second transduction. For the quantitation of 53BP1 foci and 53BP1 and TRF1 colocalization, see Fig. S1 in the supplemental material. (D) The parental and genetically complemented cell lines were transduced with control lentivirus (lanes 1 and 4) and lentiviruses for the expression of Flag-TRF2 ΔB (lanes 2 and 5) or Flag-TRF2 (lanes 3 and 6). Cells were harvested 8 days after lentivirus transduction, and genomic DNA was isolated and digested with HinfI and RsaI. Equal amounts (2 μg) of digested genomic DNA were separated by electrophoresis on a 0.8% agarose gel, followed by Southern blot analysis with a radiolabeled (TTAGGG) 3 probe. Southern blot analyses were performed on three independent samples of WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

Techniques Used: Expressing, Mutagenesis, Infection, Cell Culture, Transduction, Western Blot, Activity Assay, Staining, Standard Deviation, Quantitation Assay, Isolation, Electrophoresis, Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation

TRF2 ΔB induces telomere shortening in normal but not WS fibroblasts. Normal and WS fibroblasts expressing Flag-TRF2 ΔB (lanes 2, 5, 9, and 11) and Flag-TRF2 (lanes 3 and 6), along with normal and WS fibroblasts transduced with control viruses (lanes 1, 4, 8, and 10), were harvested 8 days after lentivirus transduction. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (TTAGGG) 3 probe. The molecular mass standards shown on right side were generated by digestion of lambda DNA with restriction endonuclease HindIII. Southern blot analyses were performed on three independent samples of normal and WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

Figure Legend Snippet: TRF2 ΔB induces telomere shortening in normal but not WS fibroblasts. Normal and WS fibroblasts expressing Flag-TRF2 ΔB (lanes 2, 5, 9, and 11) and Flag-TRF2 (lanes 3 and 6), along with normal and WS fibroblasts transduced with control viruses (lanes 1, 4, 8, and 10), were harvested 8 days after lentivirus transduction. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (TTAGGG) 3 probe. The molecular mass standards shown on right side were generated by digestion of lambda DNA with restriction endonuclease HindIII. Southern blot analyses were performed on three independent samples of normal and WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

Techniques Used: Expressing, Transduction, Electrophoresis, Agarose Gel Electrophoresis, Southern Blot, Generated, Lambda DNA Preparation, Plasmid Preparation

Expression of wild-type WRN but not enzymatically deficient WRN variants in WS fibroblasts leads to a reduction in telomeric circles, which are reformed upon the overexpression of TRF2 ΔB . (A) DNA isolated from WS fibroblasts transduced with a vector control lentivirus or a lentivirus expressing WRN was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. (B) DNA isolated from WS fibroblasts transduced with a lentivirus expressing a WRN variant lacking either exonuclease or helicase activity was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. Arrows show arcs of telomeric DNA circles. (C) DNA isolated from WRN-complemented WS fibroblasts was transduced with a control lentivirus or a lentivirus expressing TRF2 ΔB , digested with HinfI and RsaI, separated by 2DGE, and probed with a telomeric (CCCTAA) 4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate level of telomeric circles present in each sample (expressed as a percentage of the total telomeric DNA) was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

Figure Legend Snippet: Expression of wild-type WRN but not enzymatically deficient WRN variants in WS fibroblasts leads to a reduction in telomeric circles, which are reformed upon the overexpression of TRF2 ΔB . (A) DNA isolated from WS fibroblasts transduced with a vector control lentivirus or a lentivirus expressing WRN was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. (B) DNA isolated from WS fibroblasts transduced with a lentivirus expressing a WRN variant lacking either exonuclease or helicase activity was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. Arrows show arcs of telomeric DNA circles. (C) DNA isolated from WRN-complemented WS fibroblasts was transduced with a control lentivirus or a lentivirus expressing TRF2 ΔB , digested with HinfI and RsaI, separated by 2DGE, and probed with a telomeric (CCCTAA) 4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate level of telomeric circles present in each sample (expressed as a percentage of the total telomeric DNA) was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

Techniques Used: Expressing, Over Expression, Isolation, Transduction, Plasmid Preparation, Variant Assay, Activity Assay, Hybridization

3) Product Images from "Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1"

Article Title: Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1

Journal: Life Science Alliance

doi: 10.26508/lsa.201800121

$Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.$
Figure Legend Snippet: Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.

Techniques Used: Transfection, Agarose Gel Electrophoresis, Amplification, Positive Control

4) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"