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Kaneka Corp dna sequences
Dna Sequences, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dna sequences/product/Kaneka Corp
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
dna sequences - by Bioz Stars, 2020-09
92/100 stars

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High Performance Liquid Chromatography:

Article Title: Investigation of ‘Head-to-Tail’-Connected Oligoaryl N,O-Ligands as Recognition Motifs for Cancer-Relevant G-Quadruplexes
Article Snippet: .. All DNA sequences used were purchased from Eurogentec (Liège, Belgium) as synthetic oligonucleotides, purified by HPLC and dialysis. .. Graphs were constructed and data fitting was carried out in OriginPro 8.0 (Northampton, MA, USA).

Labeling:

Article Title: Aptamer-Based Molecular Recognition of Lysergamine, Metergoline and Small Ergot Alkaloids
Article Snippet: .. The following labeled DNA sequences (Eurogentec, Belgium) were used in the colorimetric reaction: 5′-ACTCATCTGTGAAGAGAAGCAGCACAGAGGTCAGATGTCCGTCAGCCCCGATCGCCATCCAGGGACTCCCCCCTATGCCTATGCGTGCTACCGTGAA-3′ (modified aptamer M3.2), 5′-TCACAGATGAGT-C3-SH-3′ (3′-SH oligo) and 5′-SH-C6-TGCTGCTTCTCT-3′ (5′-SH oligo). ..

Purification:

Article Title: Investigation of ‘Head-to-Tail’-Connected Oligoaryl N,O-Ligands as Recognition Motifs for Cancer-Relevant G-Quadruplexes
Article Snippet: .. All DNA sequences used were purchased from Eurogentec (Liège, Belgium) as synthetic oligonucleotides, purified by HPLC and dialysis. .. Graphs were constructed and data fitting was carried out in OriginPro 8.0 (Northampton, MA, USA).

Article Title: Development of microfluidic platforms for the synthesis of metal complexes and evaluation of their DNA affinity using online FRET melting assays of microfluidic platforms for the synthesis of metal complexes and evaluation of their DNA affinity using online FRET melting assays †Electronic supplementary infor
Article Snippet: .. All DNA sequences (labelled or unlabelled) were purchased RP-cartridge purified from Eurogentec (Belgium). .. 1 H and 13 C NMR spectra were recorded using a 400 MHz Bruker Avance Ultrashield NMR spectrometer (Imperial College London, Department of Chemistry, NMR Service Facility) at 296 K. Chemical shifts are given in parts per million (ppm, δ ) and referenced to the residual deuterated solvent.

Article Title: Quadruplexes in ‘Dicty’: crystal structure of a four-quartet G-quadruplex formed by G-rich motif found in the Dictyostelium discoideum genome
Article Snippet: .. All DNA sequences, listed in Table , were purchased from Eurogentec (Seraing, Belgium) with a Reverse-Phase Cartridge•Gold™ purification or from Integrated DNA Technologies (Coralville, Iowa USA) with standard desalting purification. ..

Sequencing:

Article Title: DsrR, a Novel IscA-Like Protein Lacking Iron- and Fe-S-Binding Functions, Involved in the Regulation of Sulfur Oxidation in Allochromatium vinosum ▿ ▿ †
Article Snippet: .. DsrR was detected with an antiserum raised against the following oligopeptide: H2 N-LNPRDPTYRPPSGG-CONH2 , encompassing highly immunogenic epitopes deduced from the nucleotide sequence (Eurogentec, Seraing, Belgium). .. Iron-binding assays were performed by anoxic incubation of 50 μM protein with no or 400 μM Fe(NH4 )2 (SO4 )2 in the presence of 2.5 mM dithiothreitol (DTT) at 10°C for 16 h. The samples were separated from unbound iron by passing them through a HiTrap desalting column (GE Healthcare).

Article Title: Novel Genes of the dsr Gene Cluster and Evidence for Close Interaction of Dsr Proteins during Sulfur Oxidation in the Phototrophic Sulfur Bacterium Allochromatium vinosum
Article Snippet: .. DsrE (H2 N-VNNSTRLTTPPQDDRH-CONH2 ), DsrF (H2 N-CLTRGQDTKGIGMKNF-CONH2 ), DsrH (H2 N-VNKSPFERNSLESC-CONH2 ), DsrJ (H2 N-DANRNPQPIDQPDQFC-CONH2 ), DsrO (H2 N-SQRLREIPSRQIREDL-CONH2 ), DsrK (H2 N-CDLDDPNEEEETDEAA-CONH2 ), DsrN (H2 N-ADVEMCDRPQGRGYVR-CONH2 and H2 N-TVRRGTGIDGSHDGIV-CONH2 , simultaneously), and DsrL (H2 N-MATSSDEMKMKPTWRC-CONH2 ) were detected with antisera raised against the given oligopeptides, comprising highly immunogenic epitopes deduced from the nucleotide sequence (Eurogentec, Seraing, Belgium). ..

Article Title: New α- and SIN γ-retrovectors for safe transduction and specific transgene expression in pancreatic β cell lines
Article Snippet: .. We replaced part of the Ψ region, the internal SF promoter (U3 region from the SFFV retrovirus) and the GFP sequence by a synthetically synthetized DNA sequence (Eurogentec) comprising the same part of the Ψ region, the RIP 405 promoter [ , ], and a new MCS. .. This linker was digested by Bsp120I and XhoI (all the restrictions enzymes were purchased from ThermoFischer or New England Biolabs) and introduced into pSERS11 SF GFP pre-digested by Bsp120I and SalI (see Fig. for the location of the relevant restriction sites). pSERS SF MCS was next constructed by taking a fragment containing the MCS from pSERS RIP MCS through a ClaI and Van91I digestion (Fig. ) which was then cloned by the same enzymes into pSERS11 SF GFP pre (which removed GFP).

other:

Article Title: pH-driven conformational switch between non-canonical DNA structures in a C-rich domain of EGFR promoter
Article Snippet: DNA sequences were provided lyophilized from Eurogentec (Belgium).

Modification:

Article Title: Aptamer-Based Molecular Recognition of Lysergamine, Metergoline and Small Ergot Alkaloids
Article Snippet: .. The following labeled DNA sequences (Eurogentec, Belgium) were used in the colorimetric reaction: 5′-ACTCATCTGTGAAGAGAAGCAGCACAGAGGTCAGATGTCCGTCAGCCCCGATCGCCATCCAGGGACTCCCCCCTATGCCTATGCGTGCTACCGTGAA-3′ (modified aptamer M3.2), 5′-TCACAGATGAGT-C3-SH-3′ (3′-SH oligo) and 5′-SH-C6-TGCTGCTTCTCT-3′ (5′-SH oligo). ..

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    Kaneka Corp recombinant untagged human hsf2bp
    <t>HSF2BP</t> inhibits homologous recombination in the Fanconi anemia pathway HSF2BP inhibits HR in the FA pathway ( A ) HSF2BP does not inhibit lesion unhooking in Xenopus egg extract. Prelabeled pICL was replicated in Xenopus egg extracts, replication products were isolated, digested by HincII, and separated on a denaturing agarose gel [ Fig S4B ] . The decline of the X-structures was quantified and plotted. See scheme in [ Fig S4A ] , main text and Methods for detailed description. ( B ) HSF2BP does not inhibit translesion synthesis in Xenopus egg extract. pICL was replicated in Xenopus egg extracts in the presence of 32P-lll-dCTP, replication products were isolated, digested by HincII, or HincII and SapI, and separated on a denaturing agarose gel [ Fig S4D ] . Extension products were quantified and plotted. Scheme of the assay in [ Fig S4C ] . ( C ) Inhibition of HR intermediate formation by HSF2BP revealed by 2D gel electrophoresis. pICL was replicated in Xenopus egg extracts in the presence of 32P- -dCTP, replication products were isolated, digested by HincII, and analyzed by 2D gel electrophoresis. ( D ) Stable RAD51 accumulation on pICL during repair is abrogated by HSF2BP wildtype, but not the R200T mutant. The pICL plasmid was pulled down by incubation with streptavidin beads coated with biotinylated LacI. Presence of bound RAD51 and FANCD2 was determined by immunoblotting. ( E ) Efficiency of RAD51 focus formation in HSF2BP-overproducing and control cells with or without 100 nM MMC treatment. After treatment cells were fixed, stained for immunofluorescence, mounted with DAPI and imaged using confocal microscopy. Images were scored manually for the number of foci. Data from three independent experiments is plotted, line represents average ± s.e.m., statistical significance was determined using the Kruskal-Wallis test with Dunn’s post-test. ( F ) HSF2BP inhibits BRCA2 loading at the ICL in Xenopus egg extract. pICL replication samples at the indicated time points were analyzed by BRCA2 ChIP.
    Recombinant Untagged Human Hsf2bp, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant untagged human hsf2bp/product/Kaneka Corp
    Average 84 stars, based on 1 article reviews
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    94
    Kaneka Corp sirna sequences sirna
    <t>HSF2BP</t> inhibits homologous recombination in the Fanconi anemia pathway HSF2BP inhibits HR in the FA pathway ( A ) HSF2BP does not inhibit lesion unhooking in Xenopus egg extract. Prelabeled pICL was replicated in Xenopus egg extracts, replication products were isolated, digested by HincII, and separated on a denaturing agarose gel [ Fig S4B ] . The decline of the X-structures was quantified and plotted. See scheme in [ Fig S4A ] , main text and Methods for detailed description. ( B ) HSF2BP does not inhibit translesion synthesis in Xenopus egg extract. pICL was replicated in Xenopus egg extracts in the presence of 32P-lll-dCTP, replication products were isolated, digested by HincII, or HincII and SapI, and separated on a denaturing agarose gel [ Fig S4D ] . Extension products were quantified and plotted. Scheme of the assay in [ Fig S4C ] . ( C ) Inhibition of HR intermediate formation by HSF2BP revealed by 2D gel electrophoresis. pICL was replicated in Xenopus egg extracts in the presence of 32P- -dCTP, replication products were isolated, digested by HincII, and analyzed by 2D gel electrophoresis. ( D ) Stable RAD51 accumulation on pICL during repair is abrogated by HSF2BP wildtype, but not the R200T mutant. The pICL plasmid was pulled down by incubation with streptavidin beads coated with biotinylated LacI. Presence of bound RAD51 and FANCD2 was determined by immunoblotting. ( E ) Efficiency of RAD51 focus formation in HSF2BP-overproducing and control cells with or without 100 nM MMC treatment. After treatment cells were fixed, stained for immunofluorescence, mounted with DAPI and imaged using confocal microscopy. Images were scored manually for the number of foci. Data from three independent experiments is plotted, line represents average ± s.e.m., statistical significance was determined using the Kruskal-Wallis test with Dunn’s post-test. ( F ) HSF2BP inhibits BRCA2 loading at the ICL in Xenopus egg extract. pICL replication samples at the indicated time points were analyzed by BRCA2 ChIP.
    Sirna Sequences Sirna, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna sequences sirna/product/Kaneka Corp
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    Image Search Results


    HSF2BP inhibits homologous recombination in the Fanconi anemia pathway HSF2BP inhibits HR in the FA pathway ( A ) HSF2BP does not inhibit lesion unhooking in Xenopus egg extract. Prelabeled pICL was replicated in Xenopus egg extracts, replication products were isolated, digested by HincII, and separated on a denaturing agarose gel [ Fig S4B ] . The decline of the X-structures was quantified and plotted. See scheme in [ Fig S4A ] , main text and Methods for detailed description. ( B ) HSF2BP does not inhibit translesion synthesis in Xenopus egg extract. pICL was replicated in Xenopus egg extracts in the presence of 32P-lll-dCTP, replication products were isolated, digested by HincII, or HincII and SapI, and separated on a denaturing agarose gel [ Fig S4D ] . Extension products were quantified and plotted. Scheme of the assay in [ Fig S4C ] . ( C ) Inhibition of HR intermediate formation by HSF2BP revealed by 2D gel electrophoresis. pICL was replicated in Xenopus egg extracts in the presence of 32P- -dCTP, replication products were isolated, digested by HincII, and analyzed by 2D gel electrophoresis. ( D ) Stable RAD51 accumulation on pICL during repair is abrogated by HSF2BP wildtype, but not the R200T mutant. The pICL plasmid was pulled down by incubation with streptavidin beads coated with biotinylated LacI. Presence of bound RAD51 and FANCD2 was determined by immunoblotting. ( E ) Efficiency of RAD51 focus formation in HSF2BP-overproducing and control cells with or without 100 nM MMC treatment. After treatment cells were fixed, stained for immunofluorescence, mounted with DAPI and imaged using confocal microscopy. Images were scored manually for the number of foci. Data from three independent experiments is plotted, line represents average ± s.e.m., statistical significance was determined using the Kruskal-Wallis test with Dunn’s post-test. ( F ) HSF2BP inhibits BRCA2 loading at the ICL in Xenopus egg extract. pICL replication samples at the indicated time points were analyzed by BRCA2 ChIP.

    Journal: bioRxiv

    Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

    doi: 10.1101/438945

    Figure Lengend Snippet: HSF2BP inhibits homologous recombination in the Fanconi anemia pathway HSF2BP inhibits HR in the FA pathway ( A ) HSF2BP does not inhibit lesion unhooking in Xenopus egg extract. Prelabeled pICL was replicated in Xenopus egg extracts, replication products were isolated, digested by HincII, and separated on a denaturing agarose gel [ Fig S4B ] . The decline of the X-structures was quantified and plotted. See scheme in [ Fig S4A ] , main text and Methods for detailed description. ( B ) HSF2BP does not inhibit translesion synthesis in Xenopus egg extract. pICL was replicated in Xenopus egg extracts in the presence of 32P-lll-dCTP, replication products were isolated, digested by HincII, or HincII and SapI, and separated on a denaturing agarose gel [ Fig S4D ] . Extension products were quantified and plotted. Scheme of the assay in [ Fig S4C ] . ( C ) Inhibition of HR intermediate formation by HSF2BP revealed by 2D gel electrophoresis. pICL was replicated in Xenopus egg extracts in the presence of 32P- -dCTP, replication products were isolated, digested by HincII, and analyzed by 2D gel electrophoresis. ( D ) Stable RAD51 accumulation on pICL during repair is abrogated by HSF2BP wildtype, but not the R200T mutant. The pICL plasmid was pulled down by incubation with streptavidin beads coated with biotinylated LacI. Presence of bound RAD51 and FANCD2 was determined by immunoblotting. ( E ) Efficiency of RAD51 focus formation in HSF2BP-overproducing and control cells with or without 100 nM MMC treatment. After treatment cells were fixed, stained for immunofluorescence, mounted with DAPI and imaged using confocal microscopy. Images were scored manually for the number of foci. Data from three independent experiments is plotted, line represents average ± s.e.m., statistical significance was determined using the Kruskal-Wallis test with Dunn’s post-test. ( F ) HSF2BP inhibits BRCA2 loading at the ICL in Xenopus egg extract. pICL replication samples at the indicated time points were analyzed by BRCA2 ChIP.

    Article Snippet: Anti-HSF2BP rabbit polyclonal antibodies SY8126 and SY8127 were raised against purified recombinant untagged human HSF2BP (Kaneka Eurogentec, Belgium) and used either as crude serum or after affinity purification against GST-HSF2BP immobilized on glutathione sepharose as described ( ).

    Techniques: Homologous Recombination, Isolation, Agarose Gel Electrophoresis, Translesion Synthesis, Inhibition, Two-Dimensional Gel Electrophoresis, Electrophoresis, Mutagenesis, Plasmid Preparation, Incubation, Staining, Immunofluorescence, Confocal Microscopy, Chromatin Immunoprecipitation

    ( A ) Co-immunoprecipitation of GFP-HSF2BP with N-terminal, middle, and C-terminal fragments of BRCA2. HeLa cells stably producing Flag-BRCA2 (full- length, or the fragments) were electroporated with GFP- or GFP-HSF2BP expression constructs. ( B ) Multiple alignment of HSF2BP-binding domain (BRCA2-F9) from BRCA2 sequences from vertebrates (fish to mammals). Human HSF2BP was used a seed query for hmmsearch software. Matched sequence fragments were aligned, highly similar sequences were replaced by single representative (95% redundancy filter). Human and Xenopus laevis BRCA2 fragments are indicated. Sequence identifiers contain Uniport accession numbers and the residue range for the aligned fragment within BRCA2 sequence. ( C ) Co-immunoprecipitation with anti-GFP beads of Flag-BRCA2-F6 with variants of GFP-HSF2BP containing point mutations in the region required for BRCA2 interaction. HEK293T cells were transiently transfected with two expression constructs. ( D ) Size exclusion chromatograms shown in [ Fig 3E ] overlaid with protein size markers analyzed under the same conditions. ( E ) Repeat of the Xenopus egg extract ICL repair experiment shown in [ Fig 3F ] . #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations.

    Journal: bioRxiv

    Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

    doi: 10.1101/438945

    Figure Lengend Snippet: ( A ) Co-immunoprecipitation of GFP-HSF2BP with N-terminal, middle, and C-terminal fragments of BRCA2. HeLa cells stably producing Flag-BRCA2 (full- length, or the fragments) were electroporated with GFP- or GFP-HSF2BP expression constructs. ( B ) Multiple alignment of HSF2BP-binding domain (BRCA2-F9) from BRCA2 sequences from vertebrates (fish to mammals). Human HSF2BP was used a seed query for hmmsearch software. Matched sequence fragments were aligned, highly similar sequences were replaced by single representative (95% redundancy filter). Human and Xenopus laevis BRCA2 fragments are indicated. Sequence identifiers contain Uniport accession numbers and the residue range for the aligned fragment within BRCA2 sequence. ( C ) Co-immunoprecipitation with anti-GFP beads of Flag-BRCA2-F6 with variants of GFP-HSF2BP containing point mutations in the region required for BRCA2 interaction. HEK293T cells were transiently transfected with two expression constructs. ( D ) Size exclusion chromatograms shown in [ Fig 3E ] overlaid with protein size markers analyzed under the same conditions. ( E ) Repeat of the Xenopus egg extract ICL repair experiment shown in [ Fig 3F ] . #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations.

    Article Snippet: Anti-HSF2BP rabbit polyclonal antibodies SY8126 and SY8127 were raised against purified recombinant untagged human HSF2BP (Kaneka Eurogentec, Belgium) and used either as crude serum or after affinity purification against GST-HSF2BP immobilized on glutathione sepharose as described ( ).

    Techniques: Immunoprecipitation, Stable Transfection, Expressing, Construct, Binding Assay, Fluorescence In Situ Hybridization, Software, Sequencing, Transfection, Plasmid Preparation

    Overexpression of HSF2BP disrupts the Fanconi anemia pathway HSF2BP overproduction disrupts the FA pathway ( A-D ) Clonogenic survivals of HeLa cells stably transformed with HSF2BP overexpression construct or empty vector. Each experiment was repeated at least two times with three technical replicates each. Error bars designate s.e.m. ( E ) Quantification of chromatid breaks in HeLa cells stably transformed with HSF2BP expression vectors or control, with and without overnight 100 nM MMC treatment. Example metaphase from HSF2BP overproducing cell treated with MMC is shown on the right, chromosomal aberrations indicated with arrows. ( F ) ICL repair efficiency in the presence or absence of HSF2BP. pICL was replicated in Xenopus egg extract that was supplemented with purified recombinant His-tagged hHSF2BP or buffer. Replication intermediates were isolated and digested with HincII, or HincII and SapI, and separated on agarose gel. Repair efficiency was calculated and plotted. As repair kinetics and absolute efficiency is highly dependent on the egg extract preparation single experiment is plotted here, and a replica is shown in [ Fig S2J ] . #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. See also [ Fig S2K ] .

    Journal: bioRxiv

    Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

    doi: 10.1101/438945

    Figure Lengend Snippet: Overexpression of HSF2BP disrupts the Fanconi anemia pathway HSF2BP overproduction disrupts the FA pathway ( A-D ) Clonogenic survivals of HeLa cells stably transformed with HSF2BP overexpression construct or empty vector. Each experiment was repeated at least two times with three technical replicates each. Error bars designate s.e.m. ( E ) Quantification of chromatid breaks in HeLa cells stably transformed with HSF2BP expression vectors or control, with and without overnight 100 nM MMC treatment. Example metaphase from HSF2BP overproducing cell treated with MMC is shown on the right, chromosomal aberrations indicated with arrows. ( F ) ICL repair efficiency in the presence or absence of HSF2BP. pICL was replicated in Xenopus egg extract that was supplemented with purified recombinant His-tagged hHSF2BP or buffer. Replication intermediates were isolated and digested with HincII, or HincII and SapI, and separated on agarose gel. Repair efficiency was calculated and plotted. As repair kinetics and absolute efficiency is highly dependent on the egg extract preparation single experiment is plotted here, and a replica is shown in [ Fig S2J ] . #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. See also [ Fig S2K ] .

    Article Snippet: Anti-HSF2BP rabbit polyclonal antibodies SY8126 and SY8127 were raised against purified recombinant untagged human HSF2BP (Kaneka Eurogentec, Belgium) and used either as crude serum or after affinity purification against GST-HSF2BP immobilized on glutathione sepharose as described ( ).

    Techniques: Over Expression, Stable Transfection, Transformation Assay, Construct, Plasmid Preparation, Expressing, Purification, Recombinant, Isolation, Agarose Gel Electrophoresis

    ( A ) Scheme of the lesion ICL unhooking assay. ( B ) ICL unhooking assay gel quantified in [ Fig 4A ] . ( C ) Scheme of the key events during ICL repair in Xenopus egg extract and the products measured in lesion bypass assay (see [ Fig S4D ] and [ Fig 4B ] for an example of a gel and quantification. ( D ) Lesion bypass assay gel quantified in [ Fig 4B ] . ( E ) Repeat of the experiment shown in [ Fig 4C ] . ( E-H ) Efficiency of ChIP with HSF2BP, XPF, REV1 and FANCD2 antibodies from the experiment shown in [ Fig 4F ] . ( I ) Efficiency of MMC-induced (50 ng/ml overnight) FANCD2 monoubiquitination (appearance of the slower migrating L-form) in control and HSF2BP-overproducing (human or mouse, GFP-tagged or untagged) HeLa cells. Immunoblots with two different anti-FANCD2 antibodies are shown. Asterisks indicate non-specific bands.

    Journal: bioRxiv

    Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

    doi: 10.1101/438945

    Figure Lengend Snippet: ( A ) Scheme of the lesion ICL unhooking assay. ( B ) ICL unhooking assay gel quantified in [ Fig 4A ] . ( C ) Scheme of the key events during ICL repair in Xenopus egg extract and the products measured in lesion bypass assay (see [ Fig S4D ] and [ Fig 4B ] for an example of a gel and quantification. ( D ) Lesion bypass assay gel quantified in [ Fig 4B ] . ( E ) Repeat of the experiment shown in [ Fig 4C ] . ( E-H ) Efficiency of ChIP with HSF2BP, XPF, REV1 and FANCD2 antibodies from the experiment shown in [ Fig 4F ] . ( I ) Efficiency of MMC-induced (50 ng/ml overnight) FANCD2 monoubiquitination (appearance of the slower migrating L-form) in control and HSF2BP-overproducing (human or mouse, GFP-tagged or untagged) HeLa cells. Immunoblots with two different anti-FANCD2 antibodies are shown. Asterisks indicate non-specific bands.

    Article Snippet: Anti-HSF2BP rabbit polyclonal antibodies SY8126 and SY8127 were raised against purified recombinant untagged human HSF2BP (Kaneka Eurogentec, Belgium) and used either as crude serum or after affinity purification against GST-HSF2BP immobilized on glutathione sepharose as described ( ).

    Techniques: Chromatin Immunoprecipitation, Western Blot

    Mapping of BRCA2-HSF2BP interaction ( A ) Schematic of the BRCA2 protein domain structure, key BRCA2 fragments and internal deletion variant used to map the HSF2BP-BRCA2 interaction site. Red and green fills indicate, respectively, absence or presence of HSF2BP binding. ( B ) Schematic of the key HSF2BP variants used to map the residues involved in interaction with BRCA2. Boundaries between the putative coiled coil and the armadillo repeat regions are indicated. The armadillo repeat region is predicted starting at residues 100-140 with high confidence (e.g. 97% confidence for the model for A109-V334 of hHSF2BP by Phyre2 ( Mezulis et al., 2015 ) based on β-catenin structure PDB 3SLA, which has 19% sequence identity). ( C ) Interaction between BRCA2 and HSF2BP revealed by co-immunoprecipitation between key variants. HeLa cells stably expressing Flag-tagged BRCA2 variants were transiently transfected with the indicated GFP-HSF2BP expression constructs (wt – full-length wildtype, ∆ – N-terminal and internal deletions, R200T point mutant) and anti-GFP immunoprecipitation was performed. ( D ) Direct interaction between purified recombinant HSF2BP (wild-type and R200T mutant) and GST-BRCA2 fragment F6 immobilized on glutathione sepharose beads. After washing bound proteins were released by heat denaturation, PAGE-fractionated and stained with CBB. ( E ) Interaction between purified untagged hHSF2BP and BRCA2-F8 fragment studied by analytical size exclusion chromatography. Proteins were analyzed separately (solid lines) or after co-incubation (dashed line). Complex formation between BRCA2-F8 and wild-type HSF2BP leads to an increase in hydrodynamic radius and elution in earlier fractions. This does not happen with R200T mutant. ( F ) HSF2BP R200T mutant does not inhibit ICL repair. pICL was replicated in Xenopus egg extract that was supplemented with recombinant His-tagged hHSF2BP WT or R200T mutant. Replication intermediates were isolated and digested with HincII, or HincII and SapI, and separated on agarose gel. Repair efficiency was calculated and plotted. A replica of this experiment is shown in [ Fig S3E ] . #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. ( G ) HSF2BP R200T mutant does not sensitize human cells to MMC. Clonogenic survival of U2OS cells overproducing either wild-type or R200T mutant form of human HSF2BP (or transformed with empty vector) and treated with the indicated doses of MMC for 2 h one day after seeding. Data from three independent experiments is plotted, error bars indicated s.e.m.

    Journal: bioRxiv

    Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

    doi: 10.1101/438945

    Figure Lengend Snippet: Mapping of BRCA2-HSF2BP interaction ( A ) Schematic of the BRCA2 protein domain structure, key BRCA2 fragments and internal deletion variant used to map the HSF2BP-BRCA2 interaction site. Red and green fills indicate, respectively, absence or presence of HSF2BP binding. ( B ) Schematic of the key HSF2BP variants used to map the residues involved in interaction with BRCA2. Boundaries between the putative coiled coil and the armadillo repeat regions are indicated. The armadillo repeat region is predicted starting at residues 100-140 with high confidence (e.g. 97% confidence for the model for A109-V334 of hHSF2BP by Phyre2 ( Mezulis et al., 2015 ) based on β-catenin structure PDB 3SLA, which has 19% sequence identity). ( C ) Interaction between BRCA2 and HSF2BP revealed by co-immunoprecipitation between key variants. HeLa cells stably expressing Flag-tagged BRCA2 variants were transiently transfected with the indicated GFP-HSF2BP expression constructs (wt – full-length wildtype, ∆ – N-terminal and internal deletions, R200T point mutant) and anti-GFP immunoprecipitation was performed. ( D ) Direct interaction between purified recombinant HSF2BP (wild-type and R200T mutant) and GST-BRCA2 fragment F6 immobilized on glutathione sepharose beads. After washing bound proteins were released by heat denaturation, PAGE-fractionated and stained with CBB. ( E ) Interaction between purified untagged hHSF2BP and BRCA2-F8 fragment studied by analytical size exclusion chromatography. Proteins were analyzed separately (solid lines) or after co-incubation (dashed line). Complex formation between BRCA2-F8 and wild-type HSF2BP leads to an increase in hydrodynamic radius and elution in earlier fractions. This does not happen with R200T mutant. ( F ) HSF2BP R200T mutant does not inhibit ICL repair. pICL was replicated in Xenopus egg extract that was supplemented with recombinant His-tagged hHSF2BP WT or R200T mutant. Replication intermediates were isolated and digested with HincII, or HincII and SapI, and separated on agarose gel. Repair efficiency was calculated and plotted. A replica of this experiment is shown in [ Fig S3E ] . #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. ( G ) HSF2BP R200T mutant does not sensitize human cells to MMC. Clonogenic survival of U2OS cells overproducing either wild-type or R200T mutant form of human HSF2BP (or transformed with empty vector) and treated with the indicated doses of MMC for 2 h one day after seeding. Data from three independent experiments is plotted, error bars indicated s.e.m.

    Article Snippet: Anti-HSF2BP rabbit polyclonal antibodies SY8126 and SY8127 were raised against purified recombinant untagged human HSF2BP (Kaneka Eurogentec, Belgium) and used either as crude serum or after affinity purification against GST-HSF2BP immobilized on glutathione sepharose as described ( ).

    Techniques: Variant Assay, Binding Assay, Sequencing, Immunoprecipitation, Stable Transfection, Expressing, Transfection, Construct, Mutagenesis, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Size-exclusion Chromatography, Incubation, Isolation, Agarose Gel Electrophoresis, Plasmid Preparation, Transformation Assay

    HSF2BP is a member of the BRCA complex. Identification of HSF2BP as BRCA2-interacting protein ( A ) Mass spectrometry analysis of anti-GFP immunoprecipitated proteins from Brca2GFP/GFP mES labelled by SILAC. Log2-transformed SILAC ratios from two separate (label-swap) experiments are plotted. One of the SILAC states in each experiment corresponds to 1 h incubation at 42 °C, which leads to BRCA2 degradation, the other to 37 °C control. Known members of BRCA complex are highlighted and labelled. ( B ) Proteins co-immunoprecipitated in two separate experiments with HSF2BP-GFP (vertical axis) or RAD51AP1-GFP (horizontal axis) were identified by mass-spectrometry. Combined peptide intensities (Log10-transformed) are plotted. Missing intensity values representing proteins identified only in one of the anti-GFP immunoprecipitates were replaced with values imputed from normal distribution with a downshift of 3 for visualization purpose only; their ranges are demarcated with dotted lines. ( C ) Proteins co-immunoprecipitating with PALB2-GFP and RAD51AP1-GFP from mES cells labelled by SILAC and quantified by mass-spectrometry; log10 transformed intensities are plotted, missing values imputed as in (B). ( D ) Hsf2bpGFP/+ cells were irradiated with 8 Gy, fixed, mounted in DAPI-containing media, and imaged by confocal microscopy. Maximum projection of direct GFP and DAPI fluorescence from a z-stack is shown. ( E ) Hsf2bpGFP/+ cells were irradiated with 8 Gy, fixed, processed for immunofluorescent staining with anti-53BP1 antibody (red channel) and mounted with DAPI. HSF2BP-GFP fluorescence was registered directly. ( F ) Mobility of single HSF2BP-GFP and BRCA2-GFP particles in live mES cells imaged by oblique illumination microscopy and analyzed using a single particle tracking routine as described ( Reuter et al., 2014 ). Duration of mobile and immobile states under control conditions (left column) or 2 h after irradiation with 10 Gy (middle), and the difference between the two (right) are shown. ( G ) Interaction between hBRCA2 and HSF2BP revealed by anti-GFP immunoprecipitation from HEK293T cells co-expressing GPF-HSF2BP (human or mouse) and MBP-BRCA2. ( H ) Interaction between endogenous X. laevis BRCA2 and purified recombinant human (H) or X. laevis (X) HSF2BP revealed by co-immunoprecipitation from Xenopus egg extracts with or without recombinant HSF2BP added. ( I ) Immunoblot of HSF2BP in human cancer cell lines that were identified as having elevated HSF2BP mRNA levels. Whole cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-HSF2BP antibody SY8126.

    Journal: bioRxiv

    Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

    doi: 10.1101/438945

    Figure Lengend Snippet: HSF2BP is a member of the BRCA complex. Identification of HSF2BP as BRCA2-interacting protein ( A ) Mass spectrometry analysis of anti-GFP immunoprecipitated proteins from Brca2GFP/GFP mES labelled by SILAC. Log2-transformed SILAC ratios from two separate (label-swap) experiments are plotted. One of the SILAC states in each experiment corresponds to 1 h incubation at 42 °C, which leads to BRCA2 degradation, the other to 37 °C control. Known members of BRCA complex are highlighted and labelled. ( B ) Proteins co-immunoprecipitated in two separate experiments with HSF2BP-GFP (vertical axis) or RAD51AP1-GFP (horizontal axis) were identified by mass-spectrometry. Combined peptide intensities (Log10-transformed) are plotted. Missing intensity values representing proteins identified only in one of the anti-GFP immunoprecipitates were replaced with values imputed from normal distribution with a downshift of 3 for visualization purpose only; their ranges are demarcated with dotted lines. ( C ) Proteins co-immunoprecipitating with PALB2-GFP and RAD51AP1-GFP from mES cells labelled by SILAC and quantified by mass-spectrometry; log10 transformed intensities are plotted, missing values imputed as in (B). ( D ) Hsf2bpGFP/+ cells were irradiated with 8 Gy, fixed, mounted in DAPI-containing media, and imaged by confocal microscopy. Maximum projection of direct GFP and DAPI fluorescence from a z-stack is shown. ( E ) Hsf2bpGFP/+ cells were irradiated with 8 Gy, fixed, processed for immunofluorescent staining with anti-53BP1 antibody (red channel) and mounted with DAPI. HSF2BP-GFP fluorescence was registered directly. ( F ) Mobility of single HSF2BP-GFP and BRCA2-GFP particles in live mES cells imaged by oblique illumination microscopy and analyzed using a single particle tracking routine as described ( Reuter et al., 2014 ). Duration of mobile and immobile states under control conditions (left column) or 2 h after irradiation with 10 Gy (middle), and the difference between the two (right) are shown. ( G ) Interaction between hBRCA2 and HSF2BP revealed by anti-GFP immunoprecipitation from HEK293T cells co-expressing GPF-HSF2BP (human or mouse) and MBP-BRCA2. ( H ) Interaction between endogenous X. laevis BRCA2 and purified recombinant human (H) or X. laevis (X) HSF2BP revealed by co-immunoprecipitation from Xenopus egg extracts with or without recombinant HSF2BP added. ( I ) Immunoblot of HSF2BP in human cancer cell lines that were identified as having elevated HSF2BP mRNA levels. Whole cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-HSF2BP antibody SY8126.

    Article Snippet: Anti-HSF2BP rabbit polyclonal antibodies SY8126 and SY8127 were raised against purified recombinant untagged human HSF2BP (Kaneka Eurogentec, Belgium) and used either as crude serum or after affinity purification against GST-HSF2BP immobilized on glutathione sepharose as described ( ).

    Techniques: Mass Spectrometry, Immunoprecipitation, Transformation Assay, Incubation, Irradiation, Confocal Microscopy, Fluorescence, Staining, Microscopy, Single-particle Tracking, Expressing, Purification, Recombinant, SDS Page