mouse anti-brca2 op95  (Millipore)

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of full-length BRCA2-Halo (JF549) in untreated mouse ES cells imaged at 30 ms interval.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of full-length BRCA2-Halo (JF549) in mouse ES cells treated with 2Gy of ionizing radiation imaged at 30 ms interval.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of BRCA2 ΔDBDΔCTD-Halo (JF549) in untreated mouse ES cells imaged at 30 ms interval.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of BRCA2 ΔDBDΔCTD-Halo (JF549) in mouse ES cells treated with 2Gy of ionizing radiation imaged at 30 ms interval.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: ( A ) Schematic overview of full-length mouse (top) and human (bottom) BRCA2 proteins, with key domains (DBD, CTD, NLS, BRC1-8: red bars; PALB2-binding: blue bar) and tags indicated. Deletion variants are shown in the middle. Amino acid numbers are shown in blue (mouse) and red (human). Expected molecular weight decrease for the deletion variants is shown on the right. Sequence conservation and alignment between mouse and human BRCA2 DNA-binding domain (DBD) and C-terminal domain (CTD) can be found in . ( B ) Immunoblot of total protein extract from mouse embryonic stem (mES) cells probed with indicated antibodies. Asterisk shows a specific band. Validation of the cell lines by genotyping is described in . Images of the full blots are shown in . ( C–F ) Clonogenetic survivals after ionizing radiation (IR), olaparib, mitomycin C (MMC), and cisplatin treatment with the indicated doses. At 8 Gy of IR, the percentage of surviving colonies of the ΔDBD- and ΔDBDΔCTD-Halo was too low to accurately determine the survival. Error bars indicate the range of data points. n numbers in the figure indicate the number of technical replicates executed on different days. Source data and statistics are available in . ( G ) CRISPR/Cas9-based homologous recombination assay to assess the homologous recombination proficiency of the different BRCA2 mutants. mES cells were transfected with a plasmid encoding Cas9 and the specific guide RNA (gRNA) and a repair template with the self-cleaving peptide P2A and the mCherry sequence in between two homology arms. Upon proper integration of the donor sequence at the ß-actin locus, the cells expressed mCherry. 96 hr after transfection, cells were sorted and the frequency of mCherry-positive cells was measured ( , ). To correct for the difference in transfection efficiency, a plasmid expressing blue fluorescent protein (BFP2) was co-transfected. The frequency of positive cells in every experimental replicate is normalized against wild-type BRCA2-Halo cells. Every data point indicates a technical replicate (averaged from two transfections). p-values (paired two-sided t-test) compared to full-length for the deletion variants are p = 0.0186 (ΔDBD), p = 0.0291 (ΔCTD), and p = 0.0021 (ΔDBDΔCTD), respectively. Figure 1—source data 1. Excel file with the source data of the cell survival data in and HR assay in . Figure 1—source data 2. Full western blot images from .

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Binding Assay, Molecular Weight, Sequencing, Western Blot, Biomarker Discovery, CRISPR, Homologous Recombination, Transfection, Plasmid Preparation, Expressing

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: ( A ) Cartoon depicting the CRISPR/Cas9 targetting strategy, indicating the approximate position of the gRNA sequences and the targetting construct with the homology arm and targetted cassette. ( B ) Scheme displaying the expected BRCA2 locus after targeting the different BRCA2 deletion variants, showing which exons and introns are deleted. ( C ) Genotyping polymerase chain reactions (PCRs) to validate the different cell lines. The multiplex PCR also included primers annealing outside the targetted genomic region to serve as a positive control for genomic DNA amplification efficiency. Primer sequences are reported in .

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: CRISPR, Construct, Multiplex Assay, Positive Control, DNA Amplification

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: ( A ) Representative confocal images (maximum intensity projections) of BRCA2 (red) and RAD51 (green) foci in mouse embryonic stem (mES) cells fixed 2 hr after mock or 2 Gy irradiation, without pre-extraction. Scale bar, 10 µm (full images can be found at ). ( B ) Quantification of the number of BRCA2-Halo (JF646) foci per nucleus of EdU+ cells irradiated with 2Gy ionizing radiation (IR) in cells without pre-extraction; three technical replicates, at least 250 cells per condition . ( C ) Distribution of integrated BRCA2 intensity per focus. ( D ) Quantification of the number of RAD51 foci in EdU+ cells irradiated with 2 Gy IR and fixed after indicated number of hours with pre-extraction for RAD51 immunostaining. Example images and percentage of EdU+ cells per condition are shown in ; three technical replicates, at least 100 cells per condition (statistical data available in ). ( E ) Fold change of foci number with respect to untreated cells. ( F ) Integrated RAD51 intensity per focus. ( G ) Fold change in integrated intensity of RAD51 foci relative to untreated cells. Representative images are shown in . Data plotted per time point can be found in . In boxplots in ( C ) and ( F ), distribution outliers are not shown; source data is available in . Figure 2—source data 1. Excel file with exact n numbers and statistical tests of and the source data of the foci quantification. Figure 2—source data 2. Original uncropped images from . Confocal z-projection images.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Irradiation, Extraction, Immunostaining

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: ( A ) Wide-field image of an S-phase cell visualized with iRFP720-PCNA and BRCA2-HaloTag::JF549. ( B ) Example of two tracks of BRCA2-Halo showing different diffusive behavior; see also – . ( C ) Distribution of apparent diffusion coefficients of segmented tracks (tracklets) for immobile (blue), slow (yellow), and fast (red) molecules for full-length BRCA2 in untreated cells; plots for ionizing radiation (IR)-treated cells and other BRCA2 variants are shown in . ( D ) Apparent diffusion rate of fast diffusing BRCA2 tracklets for full-length BRCA2 and indicated deletion variants. p-values (two-sided t-test) comparing full-length with deletion variants (ΔDBD, ΔCTD, ΔDBDΔCTD) are, respectively, p = 0.953, p = 0.797, p = 0.593. ( E ) Immobile fraction estimated by segmentation of tracks by their immobile, slow, or fast mobility (tracklets). Fraction is defined as the percentage of tracklets per cell that are immobile. Cells were imaged between 2 and 4 hr after IR treatment. p-values (two-sided t-test) comparing -/+ IR for different variants (full-length, ΔDBD, ΔCTD, ΔDBDΔCTD) are, respectively, p = 0.08, p = 0.057, p = 0.02, p = 0.4. Merged data from two independent experiments of at least 15 cells and about 10,000 tracks per condition are shown . Percentages below the plot indicate the median immobile fraction of tracklets per condition. Figure 3—source data 1. Excel file with exact n numbers and statistical tests of and the source data of the single-molecule-tracking experiments.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Diffusion-based Assay

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: ( A ) Representative scanning force microscopy (SFM) height images of full-length and ∆CTD BRCA2 in the presence and absence of RAD51. BRCA2 ΔCTD forms rod-shaped assemblies, like full-length BRCA2, on interaction with RAD51. Rod-like assemblies are indicated by green arrows; pink arrows indicate multimeric assemblies, based on volume analyses. ( B ) Histograms showing oligomeric distribution of full-length BRCA2 and the C-terminal variants in the presence and absence of RAD51. The deletion of C-terminal region leads to lesser oligomeric forms than full-length BRCA2. All the experiments were performed twice with independent protein preparations, imaging, and analyses. The figure is plotted from one of the duplicate data sets. Both data sets can be found in . Figure 4—source data 1. Excel files with the source data of the data in and the replicate experiment.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Microscopy, Imaging

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: ( A ) Representative scanning force microscopy (SFM) height images of full-length BRCA2 and BRCA2 ΔDBD in the presence and absence of single-stranded DNA (ssDNA). Full-length BRCA2 rearranges into extended molecular assemblies on interaction with ssDNA; however, BRCA2 ΔDBD and other C-terminal constructs do not show any conformational change. Pink arrows indicate the oligomeric volume of the particle with respect to the BRCA2 monomer. ( B ) Distribution of full-length BRCA2 and the C-terminal deletion constructs with respect to their oligomerization and solidity. Full-length BRCA2 rearranges to form extended dimers and tetramers on interaction with ssDNA, whereas the deletion constructs do not show any change in their distribution. All the experiments were performed twice with independent protein preparations, imaging, and analyses. The figure is plotted from one of the duplicate data sets. Both data sets can be found in . Figure 5—source data 1. Excel files with the source data of the data in and the replicate experiment.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Microscopy, Construct, Imaging

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: A plasmid expressing BFP2 was co-introduced for the selection of successfully transfected cells. The percentage of transfected cells (BFP+) that express mCherry targetted to the β-actin cells was quantified. Typical results are shown for full-length BRCA2-Halo and for BRCA2-Halo ΔDBDΔCTD.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Plasmid Preparation, Expressing, Selection, Transfection

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: ( A ) Confocal images (maximum intensity projection) of mouse embryonic stem (mES) cells producing the indicated variants of BRCA2 immunostained for RAD51 after pre-extraction and pulse-labeling with EdU click chemistry to reveal S-phase cells. ( B ) Fraction of EdU-positive (S-phase) mES cells revealed as shown in panel ( A ) at different time points after irradiation with 2 Gy. Average from three experiments, bars indicate SEM. Source data is available in . ( C ) Data from replotted for all variants per time point. ( D ) Confocal images of BRCA2-Halo::JF646. ( E ) Data from replotted for all variants per time point.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Extraction, Labeling, Irradiation

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: ( A ) Wide-field images of cells from the different BRCA2 variants (like in ), showing iRFP720-PCNA in magenta and BRCA2-Halo::JF549 in green. ( B ) Histograms of the apparent diffusion constant estimated for every tracklet. Tracks are segmented in fast (red), slow (yellow), and immobile (blue) tracklets using the DL-MSS software. The numbers above the plots indicate the average fractions +/- standard deviation estimated from fractions per cell.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Diffusion-based Assay, Software, Standard Deviation

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: All the BRCA2 samples, full-length and the deletion constructs, show a similar rearrangement into globular and monomeric assemblies.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Construct

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Control experiment showing representative scanning force microscopy (SFM) height images of full-length BRCA2 ± ssDNA in the absence of spermidine, showing that the conformational change observed on interaction with single-stranded DNA (ssDNA) is not an artifact due to presence of spermidine, which is used to facilitate adsorption of DNA on the mica surface for SFM imaging.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Control, Microscopy, Adsorption, Imaging

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Summary of results of the in vivo assays in this study.

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: In Vivo, Diffusion-based Assay

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti BRCA2 , Calbiochem , OP95 , WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500).

Techniques: Suspension, Recombinant, Plasmid Preparation, Expressing, Modification, Knock-In, Construct, Sequencing, Mutagenesis, Transfection, Software

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of full-length BRCA2-Halo (JF549) in untreated mouse ES cells imaged at 30 ms interval.

Article Snippet: Peak elution fractions were checked by western blot using mouse monoclonal anti-BRCA2 (OP95-Calbiochem) as the primary antibody (1:500) and sheep anti-mouse HRP (1:2000) (Jackson ImmunoResearch) as the secondary antibody.

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of full-length BRCA2-Halo (JF549) in mouse ES cells treated with 2Gy of ionizing radiation imaged at 30 ms interval.

Article Snippet: Peak elution fractions were checked by western blot using mouse monoclonal anti-BRCA2 (OP95-Calbiochem) as the primary antibody (1:500) and sheep anti-mouse HRP (1:2000) (Jackson ImmunoResearch) as the secondary antibody.

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of BRCA2 ΔDBDΔCTD-Halo (JF549) in untreated mouse ES cells imaged at 30 ms interval.

Article Snippet: Peak elution fractions were checked by western blot using mouse monoclonal anti-BRCA2 (OP95-Calbiochem) as the primary antibody (1:500) and sheep anti-mouse HRP (1:2000) (Jackson ImmunoResearch) as the secondary antibody.

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet: Single-molecule recording of BRCA2 ΔDBDΔCTD-Halo (JF549) in mouse ES cells treated with 2Gy of ionizing radiation imaged at 30 ms interval.

Article Snippet: Peak elution fractions were checked by western blot using mouse monoclonal anti-BRCA2 (OP95-Calbiochem) as the primary antibody (1:500) and sheep anti-mouse HRP (1:2000) (Jackson ImmunoResearch) as the secondary antibody.

Techniques:

Journal: eLife

Article Title: Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

doi: 10.7554/eLife.67926

Figure Lengend Snippet:

Article Snippet: Peak elution fractions were checked by western blot using mouse monoclonal anti-BRCA2 (OP95-Calbiochem) as the primary antibody (1:500) and sheep anti-mouse HRP (1:2000) (Jackson ImmunoResearch) as the secondary antibody.

Techniques: Suspension, Recombinant, Plasmid Preparation, Expressing, Modification, Knock-In, Construct, Sequencing, Mutagenesis, Transfection, Software

Journal: PLoS ONE

Article Title: Ex vivo assays to predict enhanced chemosensitization by hyperthermia in urothelial cancer of the bladder

doi: 10.1371/journal.pone.0209101

Figure Lengend Snippet: A) Immunoblots of samples treated with or without hyperthermia, probed for BRCA2 and PARP-1 as a loading control. B) RT112 cells were treated with or without hyperthermia and subsequently irradiated with the indicated dose. Cells were then incubated at 37°C, and fixed at indicated time-points afterward irradiation. EdU was present for one hour prior to fixation to identify S phase cells. Cells were stained for EdU and RAD51. Pictures are a maximum projection of a Z-stack, and the dotted lines denote the perimeter of EdU-positive nuclei. C) The 2-dimensional graph expresses a quantified classification of RAD51 foci. Each dot in the graph represents one cell, plotted based on the number of RAD51 foci in its nucleus (y axis) and their integrated density (x axis); a derivate of intensity and area per nucleus. Cells without foci are presented in the embedded figure noting ‘0 foci’. D) and E) As panel B and C, but for T24 cells.

Article Snippet: Proteins were detected with primary antibodies mouse-anti-BRCA2 (OP95, Calbiochem, 1:1000) or mouse-anti-PARP-1 (Alexa 1:1000) and HRP-conjugated Sheep anti-mouse IgG (H+L) (1:2000, Jackson ImmunoResearch) as secondary antibody.

Techniques: Western Blot, Control, Irradiation, Incubation, Staining