mouse α-brca2 (ab-1)  (Millipore)

Journal: bioRxiv

Article Title: EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

doi: 10.1101/2023.02.24.529205

Figure Lengend Snippet: (A) A published gene essentiality screen in BRCA2-proficient and -deficient RPE1 cells ( Adam et al. , 2021 ) was mined to extract the CCA scores for the indicated genes. A higher CCA score indicates a unique essentiality in BRCA2-deficient cells compared to proficient cells. Dashed line indicates the cut-off for a significant CCA score (based on Adam et al. , 2021 ). (B) DLD1 WT and BRCA2 -/- cells were infected with empty vector (CTRL) or EXO1 -targeting sgRNA and viability was measured using CellTiter-Glo (n=3, mean+SD, paired t-test). (C) Lysates of the indicated DLD1 cells indicated in analysed by western blotting. Samples were run on the same gel, dashed line indicates removal of non-relevant lanes post-imaging. (D) H1299 cells carrying a doxycycline-inducible BRCA2 shRNA were transduced with an AAVS1-targeting (CTRL) or EXO1 -targeting sgRNA, followed by a clonogenic survival assay in absence or presence of 10 μg/mL doxycycline (n=3, mean+SD, paired t-test). (E) Western blot analysis of the lysates of the H1299 TetOn sh BRCA2 cells studied in .

Article Snippet: The following primary antibodies were used for western blotting: Mouse α 53BP1 (BD 612522; 1:1,500), Rabbit α BLM (Abcam ab2179; 1:2,000), Mouse α BRCA1 (Merck OP92; 1:1,000), Rabbit α BRCA1 (Millipore 07-434; 1:2,000), Mouse α BRCA2 (Merck OP95; 1:1,000), Mouse α CTIP (Millipore MABE1060; 1:1,000), Rabbit α EXO1 (Abcam ab95068; 1:1,000), Rabbit α EXO1 (Millipore ABE1354; 1:1,000), Mouse α GFP (Sigma #11814460001; 1:1000), Rabbit α MRE11 (1:3,000; de Jager et al., 2001), Mouse α TUBULIN (Sigma T6199; 1:5,000), and Mouse α RAD52 (Santa-Cruz sc-365341; 1:100).

Techniques: Infection, Plasmid Preparation, Western Blot, Imaging, shRNA, Transduction, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

doi: 10.1101/2023.02.24.529205

Figure Lengend Snippet: (A) RPE1 hTERT TP53 -/- BRCA1 -/- cells expressing Cas9, either TP53BP1 +/+ or TP53BP1 -/- , were transduced with an AAVS1-targeting (CTRL) or EXO1 -targeting sgRNA, followed by a clonogenic survival assay (n=3, mean+SD, **p<0.01, paired t-test). Western blot of lysates shown in supplemental figure 2A. (B) Indicated RPE1 cell lines were incubated with EdU and PARGi for 30 minutes, followed by IF microscopy to analyse PAR formation in EdU-positive cells. A representative of two independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney). (C) As in , but for BRCA1 -mutated MDA-MB-436 cells, either WT or reconstituted with BRCA1 cDNA, infected with empty vector (CTRL) or EXO1 -targeting sgRNA. A representative of three independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney). (D) As in , but for DLD1 cells, either WT or BRCA2 -/- . A representative of three independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney).

Article Snippet: The following primary antibodies were used for western blotting: Mouse α 53BP1 (BD 612522; 1:1,500), Rabbit α BLM (Abcam ab2179; 1:2,000), Mouse α BRCA1 (Merck OP92; 1:1,000), Rabbit α BRCA1 (Millipore 07-434; 1:2,000), Mouse α BRCA2 (Merck OP95; 1:1,000), Mouse α CTIP (Millipore MABE1060; 1:1,000), Rabbit α EXO1 (Abcam ab95068; 1:1,000), Rabbit α EXO1 (Millipore ABE1354; 1:1,000), Mouse α GFP (Sigma #11814460001; 1:1000), Rabbit α MRE11 (1:3,000; de Jager et al., 2001), Mouse α TUBULIN (Sigma T6199; 1:5,000), and Mouse α RAD52 (Santa-Cruz sc-365341; 1:100).

Techniques: Expressing, Transduction, Clonogenic Cell Survival Assay, Western Blot, Incubation, Microscopy, MANN-WHITNEY, Infection, Plasmid Preparation

Journal: bioRxiv

Article Title: EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

doi: 10.1101/2023.02.24.529205

Figure Lengend Snippet: (A) Results of a gene essentiality screen in BRCA1-proficient and -deficient cells ( Adam et al. , 2021 ) were mined to extract the CCA scores for the indicated genes. A higher CCA score indicates a unique essentiality in BRCA1-deficient cells compared to proficient cells. Dashed line indicates the cut-off for a significant CCA score (based on Adam et al. , 2021 ). (B) Indicated RPE1 cells were transfected with a control (siCTRL) or EXO1 -targeting siRNA, followed by treatment with IR and subsequent IF microscopy to analyse either RAD51 or RAD52 foci formation. Left panel shows quantification (n=3, mean+SD, *p<0.05, paired t-test) and right panel shows representative microscopy images of eGFP-RAD52 foci. Western blot of lysates shown in supplemental figure 3A. (C) Clonogenic survival assay of Cas9-expressing RPE1 hTERT TP53 -/- BRCA1 -/- cells that were transduced to express the indicated sgRNAs (n=3, mean+SD, *p<0.05, **p<0.01, one-way ANOVA, post-hoc Dunnett’s, compared to CTRL). Western blot of lysates shown in supplemental figure 3D. (D) HEK 293T cells carrying the DSB-Spectrum_V3 reporter, either WT or EXO1 -/- , were transfected with indicated siRNAs, followed by a second round of transfection with a Cas9 cDNA and BFP sgRNA targeting the reporter locus. Next, cells were analysed by flow cytometry to quantify repair by the indicated pathways (n=4, mean±SEM, *p<0.05, ratio paired t-test). Western blot of lysates shown in supplemental figure 3E. (E) Nuclear γH2AX intensity in S-phase (EdU + ) cells analysed by IF microscopy in BRCA2 -/- cells infected with empty vector or sgEXO1 . A representative of two independent experiments is shown, black line indicates median.

Article Snippet: The following primary antibodies were used for western blotting: Mouse α 53BP1 (BD 612522; 1:1,500), Rabbit α BLM (Abcam ab2179; 1:2,000), Mouse α BRCA1 (Merck OP92; 1:1,000), Rabbit α BRCA1 (Millipore 07-434; 1:2,000), Mouse α BRCA2 (Merck OP95; 1:1,000), Mouse α CTIP (Millipore MABE1060; 1:1,000), Rabbit α EXO1 (Abcam ab95068; 1:1,000), Rabbit α EXO1 (Millipore ABE1354; 1:1,000), Mouse α GFP (Sigma #11814460001; 1:1000), Rabbit α MRE11 (1:3,000; de Jager et al., 2001), Mouse α TUBULIN (Sigma T6199; 1:5,000), and Mouse α RAD52 (Santa-Cruz sc-365341; 1:100).

Techniques: Transfection, Microscopy, Western Blot, Clonogenic Cell Survival Assay, Expressing, Flow Cytometry, Infection, Plasmid Preparation

Journal: bioRxiv

Article Title: EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

doi: 10.1101/2023.02.24.529205

Figure Lengend Snippet: (A) Model of the mechanism causing synthetic lethality between BRCA1-deficiency and EXO1 loss. (B) Whole genome sequencing data of pan cancer tumour samples ( Martínez-Jiménez et al. , 2022 ) was analysed to quantify the number of genetic scars indicative of DSB-repair by SSA, here defined as deletions flanked by homologous sequences of >10bp. CHORD analysis was used to classify samples as HR-proficient or HR-deficient, either BRCA1-type or BRCA2-type ( Nguyen et al. , 2020 ) (****p<0.0001, kolmogornov-smirnov). (C) Tumour samples from a pan-cancer cohort were binned based on SSA scar count, and the EXO1 expression was plotted for each tumour sample. (D) EXO1 expression levels in BRCA1 WT or BRCA1 mutant pan-cancer tumour samples.

Article Snippet: The following primary antibodies were used for western blotting: Mouse α 53BP1 (BD 612522; 1:1,500), Rabbit α BLM (Abcam ab2179; 1:2,000), Mouse α BRCA1 (Merck OP92; 1:1,000), Rabbit α BRCA1 (Millipore 07-434; 1:2,000), Mouse α BRCA2 (Merck OP95; 1:1,000), Mouse α CTIP (Millipore MABE1060; 1:1,000), Rabbit α EXO1 (Abcam ab95068; 1:1,000), Rabbit α EXO1 (Millipore ABE1354; 1:1,000), Mouse α GFP (Sigma #11814460001; 1:1000), Rabbit α MRE11 (1:3,000; de Jager et al., 2001), Mouse α TUBULIN (Sigma T6199; 1:5,000), and Mouse α RAD52 (Santa-Cruz sc-365341; 1:100).

Techniques: Sequencing, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

doi: 10.1101/2022.12.28.522061

Figure Lengend Snippet: A) Schematic of experimental approach combining fluorescence microscopy, a microfluidic flow-cell, and optical-trapping, as well as the micromanipulation used to capture and image BRCA2 on individual DNA molecules. B) Illustration of the gapped λ DNA generated through in vitro recombination of circular ssDNA with an engineered λ DNA. C) Schematic of experimental protocol: each molecule of gapped λ DNA was captured and micromanipulated between two beads held in separately controllable optical traps. The molecule was moved between solutions in a six-channel flow cell, and successively incubated in a solution containing BRCA2. D) Cartoon and microscopic image of a single-molecule of gapped λ DNA ( left, stained with YOYO-1, cyan ) that was destained and then successively incubated with BRCA2 (5 nM) plus α-BRCA2 and α-IgG AF546 . Montage shows BRCA2 ( magenta ) binding to the gapped λ DNA at increasing time intervals. E) Cartoon representation of the gapped λ DNA between two beads ( top ) and histogram ( middle ) of binding positions of BRCA2 (number of foci, N =60). Each data point is also plotted as a single tick ( bottom ) where the semi-transparent box represents the standard error associated with assigning position owing to the optical resolution of the microscope. Gray bars represent the 10-90 th percentile range of the 5’- and 3’-termined junctions ( N =98).

Article Snippet: Experiments designed to visualize BRCA2 binding, either in the presence or absence of fluorescent RAD51, contained 1.5 µg/mL α-MBP AF546 (Abcam, labelled with Alexa Fluor 546, Molecular Probes) or 1.5 µg/mL mouse α-BRCA2 (Ab-1, Millipore) plus 1-2 µg/mL goat α-mouse IgG AF546 (Molecular Probes) as indicated in the figure legends.

Techniques: Fluorescence, Microscopy, Micromanipulation, Generated, In Vitro, Incubation, Staining, Binding Assay

Journal: bioRxiv

Article Title: BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

doi: 10.1101/2022.12.28.522061

Figure Lengend Snippet: Montage of a single gapped λDNA molecule held between two optically trapped polystyrene beads in a 6-channel flow cell as described in . The molecule was imaged in Channel 5 and iteratively dipped into Channel 6, containing BRCA2 (purple, α-MBP AF546 ), by moving the molecule ( down, then up ) perpendicular to the flow (from left to right ) between images in the montage. The BRCA2 focus highlighted between 32 s and 64 s slides upwards towards the junction between the dsDNA and RPA-coated ssDNA. At the end of the movie, the gapped λDNA was re-stained in Channel 3, whereupon the less stable BRCA2-dsDNA complexes dissociated.

Article Snippet: Experiments designed to visualize BRCA2 binding, either in the presence or absence of fluorescent RAD51, contained 1.5 µg/mL α-MBP AF546 (Abcam, labelled with Alexa Fluor 546, Molecular Probes) or 1.5 µg/mL mouse α-BRCA2 (Ab-1, Millipore) plus 1-2 µg/mL goat α-mouse IgG AF546 (Molecular Probes) as indicated in the figure legends.

Techniques: Staining

Journal: bioRxiv

Article Title: BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

doi: 10.1101/2022.12.28.522061

Figure Lengend Snippet: A) Schematic of a single molecule of gapped λ DNA attached at each end to a PEG-coated surface via neutravidin. The dsDNA region was initially visualized using SYTOX Orange ( red ), which was subsequently dissociated upon the addition of binding buffer and fluorescein-RAD51 ( green ). B) In the absence of RPA, fluorescent RAD51 rapidly filled the ssDNA region. C) When RPA was present, RAD51 binding was slower and punctate. D) Comparison of the lag time in the absence (black filled symbols) or presence (black open symbols) of RPA measured using TIRF microscopy. Lag times in the presence of BRCA2 were measured using optical trapping ( see text ) and are shown in red symbols for comparison. Lines represent the arithmetic mean and error bars represent standard deviation. No RPA: 5±3 ( N =111); +RPA: 78±40 ( N =20); +RPA/BRCA2: 27±11 ( N =22). Scale bar in panels b and c is 2 µm.

Article Snippet: Experiments designed to visualize BRCA2 binding, either in the presence or absence of fluorescent RAD51, contained 1.5 µg/mL α-MBP AF546 (Abcam, labelled with Alexa Fluor 546, Molecular Probes) or 1.5 µg/mL mouse α-BRCA2 (Ab-1, Millipore) plus 1-2 µg/mL goat α-mouse IgG AF546 (Molecular Probes) as indicated in the figure legends.

Techniques: Binding Assay, Microscopy, Standard Deviation

Journal: bioRxiv

Article Title: BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

doi: 10.1101/2022.12.28.522061

Figure Lengend Snippet: A) Schematic of optical-trap experiments designed to visualize nucleation of RAD51 on gapped λ DNA in the absence or presence of BRCA2. B) A single molecule of gapped λ DNA with bound RPA held between two optical traps and co-incubated with 100 nM RAD51 (green) and 5 nM BRCA2 (red, α-MBP AF546 ). C) Cartoon of the gapped λ DNA ( top ) and histogram ( middle ) showing positions of all RAD51 nucleation events either alone ( green ) ( N =18) or when co-incubated with BRCA2 ( red ) in the absence of antibody ( N =28). Positions of individual foci are plotted ( bottom ) relative to position of the 5’- and 3’-termined junctions. The transparent box around each dash represents the standard error owing to the optical resolution of our microscope. Gray bars represent the 10-90 th percentile range of the 5’- and 3’-termined junctions ( N =98). D) Bar plot of the number of RAD51 nucleation events in the absence ( green ) or presence of BRCA2 ( red ) observed in the regions nearest the 3’-junction, clearly in the middle of the RPA-coated region, or near the 5’-junction. The odds ratios and p-values were calculated using Fisher’s exact test.

Article Snippet: Experiments designed to visualize BRCA2 binding, either in the presence or absence of fluorescent RAD51, contained 1.5 µg/mL α-MBP AF546 (Abcam, labelled with Alexa Fluor 546, Molecular Probes) or 1.5 µg/mL mouse α-BRCA2 (Ab-1, Millipore) plus 1-2 µg/mL goat α-mouse IgG AF546 (Molecular Probes) as indicated in the figure legends.

Techniques: Incubation, Microscopy

Journal: bioRxiv

Article Title: BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

doi: 10.1101/2022.12.28.522061

Figure Lengend Snippet: A) The cumulative frequency of RAD51 nucleation measured by optical trapping is plotted as a function of increasing incubation time at varying concentrations of RAD51 in the absence or B) presence of BRCA2. Inset shows a zoomed in scale. The solid lines represent non-linear fitting to a single-exponential rate equation: 50 nM: t 1/2 =110±10 s, 100 nM: t 1/2 =47±4 s, 200 nM: t 1/2 =8±1 s, 300 nM: t 1/2 =11±4 s (std. error). The open symbols and cyan dashed line represent the kinetics of BRCA2 binding in the absence of RAD51 as measured in and is shown as a comparison to the kinetics of RAD51 nucleation ( see text ). C) The arithmetic mean nucleation time plotted as a function of increasing RAD51 concentration in the absence: 50 nM: 179±41 s ( N =10), 100 nM: 76±16 s ( N =14), 200 nM: 38±23 s ( N =4), 300 nM: 23±4 s ( N =4), 400 nM: 30±6 s ( N =6), or presence of BRCA2: 50 nM: 24±10 s ( N =7), 100 nM: 21±2 s ( N =46), 400 nM: 15±6 s ( N =7). The black curve represents a fit to the power law ( J = k [RAD51] n ) where n = 1.5±0.3 (std. error). The cyan symbol represents the half-time for BRCA2 binding in the absence of RAD51, measured with α-BRCA2 plus α-IgG AF546 : 30±6 s ( N =6). The red line is for visual purposes only; gray symbol is the rate in the absence of RPA: 5.2±0.3 s ( N =111). Error and error bars are standard error of the mean, and if not visible, are smaller than the symbol. D) Model of BRCA2-mediated RAD51 nucleation on RPA-coated ssDNA.

Article Snippet: Experiments designed to visualize BRCA2 binding, either in the presence or absence of fluorescent RAD51, contained 1.5 µg/mL α-MBP AF546 (Abcam, labelled with Alexa Fluor 546, Molecular Probes) or 1.5 µg/mL mouse α-BRCA2 (Ab-1, Millipore) plus 1-2 µg/mL goat α-mouse IgG AF546 (Molecular Probes) as indicated in the figure legends.

Techniques: Incubation, Binding Assay, Concentration Assay

Journal: PLoS Genetics

Article Title: ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination

doi: 10.1371/journal.pgen.1003667

Figure Lengend Snippet: ( A ) A549 tumor cells treated with BRCA2 siRNA were irradiated with 1 Gy (0.5 h) or 2 Gy (8 h) and immunostained with the indicated antibodies. Using EdU and cell cycle markers to distinguish G1- from G2-phase cells , focal intensities of pATM were measured using ImageJ software (see ). BRCA2 siRNA was used in this analysis to accumulate resected DSBs. ( B ) 2BN hTert (XLF-deficient) human fibroblasts were analyzed 2 h post IR with 1 Gy. Cells were stained against γH2AX and RAD51 or pATM and RAD51, and γH2AX or pATM focal intensities were measured at RAD51-foci-positive or RAD51-foci-negative foci. XLF-deficient cells were used in this analysis to prevent repair of EC DSBs during the time needed for resection of HC DSBs. ( C ) 82-6 hTert (wt) and F02-98 hTert (ATR-deficient) human fibroblasts were stained against γH2AX and RAD51 at 2 h post 1 Gy, and γH2AX focal intensities were measured as in (B). ( D ) 2BN hTert (XLF-deficient) human fibroblasts were stained against γH2AX and RAD51 at 2 h post 1 Gy, and γH2AX focal intensities were measured as in (B). Since both DNA-PK and ATM can phosphorylate γH2AX, cells were treated with DNA-PK inhibitor under all conditions. Inhibitors were added 1 h post IR, a time sufficient to allow for ATM-dependent resection and RAD51 loading (see ). In (A–D), at least 300 foci from 3 independent experiments were analyzed for each point. Box plots were used with a maximum whisker-length of 1.5-fold the inter-quartile range; the lower and upper “x” indicates the 1% or 99% margin of the data range, respectively.

Article Snippet: Primary antibodies used were: rabbit-α-pATM at 1∶1000 (Epitomics); rabbit-α-pKAP-1 (S824) at 1∶10000 (Epitomics); rabbit-α-KAP-1 at 1∶1000 (abcam); mouse-α-BRCA2 at 1∶1000 (Cell signaling); rabbit-α-GAPDH at 1∶1000 (Santa Cruz); mouse-α-γH2AX at 1∶1000 (Millipore); mouse-α-H3 at 1∶1000 (abcam); mouse-α-RPA2 at 1∶1000 (Calbiochem); rabbit-α-pRPA2 (S4/8) at 1∶10000 (Bethyl).

Techniques: Irradiation, Software, Staining, Whisker Assay

Journal: PLoS Genetics

Article Title: ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination

doi: 10.1371/journal.pgen.1003667

Figure Lengend Snippet: ( A ) γH2AX foci and PCC analysis in G2-irradiated 82-6 hTert (wt) and HSC62 hTert (BRCA2-deficient) human fibroblasts. ( B ) RPA and RAD51 foci analysis in G2-irradiated A549 tumor cells. ( C ) Endogenous KAP-1 and BRCA2 was depleted in HeLa tumor cells by siRNA, and cells were transfected with GFP-tagged and siRNA-resistant empty (GFP), wt or mutated (phospho-mutant S824A or phospho-mimic S824D) KAP-1 plasmids. γH2AX foci were analyzed in GFP-positive G2-irradiated cells. EdU and cell cycle markers were used to distinguish G2- from S- and G1-phase cells . In (A), (B) and (C), foci numbers or PCC breaks from unirradiated cells were subtracted. At least 40 cells or PCC spreads were analyzed per data point and experiment (mean ± SEM from ≥3 experiments). KAP-1 and BRCA2 depletion in this and subsequent experiments was highly efficient (>90% as assessed by Western blotting). P values were obtained by t -test and represent a comparison of all cells analyzed in the indicated cell populations (***: p<0.001).

Article Snippet: Primary antibodies used were: rabbit-α-pATM at 1∶1000 (Epitomics); rabbit-α-pKAP-1 (S824) at 1∶10000 (Epitomics); rabbit-α-KAP-1 at 1∶1000 (abcam); mouse-α-BRCA2 at 1∶1000 (Cell signaling); rabbit-α-GAPDH at 1∶1000 (Santa Cruz); mouse-α-γH2AX at 1∶1000 (Millipore); mouse-α-H3 at 1∶1000 (abcam); mouse-α-RPA2 at 1∶1000 (Calbiochem); rabbit-α-pRPA2 (S4/8) at 1∶10000 (Bethyl).

Techniques: Irradiation, Transfection, Mutagenesis, Western Blot, Comparison

Journal: PLoS Genetics

Article Title: ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination

doi: 10.1371/journal.pgen.1003667

Figure Lengend Snippet: ( A ) γH2AX foci and PCC analysis in G2-irradiated 82-6 hTert (wt) and 2BN hTert (XLF-deficient) human fibroblasts. ( B ) γH2AX foci and PCC analysis in G2-irradiated 82-6 hTert (wt) human fibroblasts treated with PARPi 0.5 h prior to IR. ( C ) PCC analysis from G2-irradiated HSC62 hTert (BRCA2-deficient) human fibroblasts treated with PARPi as in (B). ( D ) γH2AX foci analysis in G2-irradiated 82-6 hTert (wt) and HSC62 hTert (BRCA2-deficient) human fibroblasts. ( E–G ) Chromatid fusions and breaks in G2-irradiated mitotic HeLa tumor cells at 8 h post 2 Gy. Cells were treated with caffeine and colcemid at 5 h post IR to abolish the G2 checkpoint and collected in mitosis. Chromosomes were stained with Giemsa (panel E) or analyzed by FISH with probes specific for chromosomes 1 (red), 2 (green) and 4 (yellow) (panel F) or to chromosomes 18 and 19 (panel G). Foci numbers, chromatid breaks and fusions from unirradiated cells were subtracted. For panels A–E, at least 40 cells or 40 PCC/mitotic spreads were analyzed per data point and experiment (mean ± SEM from ≥3 experiments). For panel G, 50 mitotic spreads were analyzed per data point and experiment (mean ± SEM from ≥2 experiments). N.d. indicates that no chromatid fusions were observed under these conditions. P values were obtained by t -test and represent a comparison of all cells analyzed in the indicated cell populations (***: p<0.001).

Article Snippet: Primary antibodies used were: rabbit-α-pATM at 1∶1000 (Epitomics); rabbit-α-pKAP-1 (S824) at 1∶10000 (Epitomics); rabbit-α-KAP-1 at 1∶1000 (abcam); mouse-α-BRCA2 at 1∶1000 (Cell signaling); rabbit-α-GAPDH at 1∶1000 (Santa Cruz); mouse-α-γH2AX at 1∶1000 (Millipore); mouse-α-H3 at 1∶1000 (abcam); mouse-α-RPA2 at 1∶1000 (Calbiochem); rabbit-α-pRPA2 (S4/8) at 1∶10000 (Bethyl).

Techniques: Irradiation, Staining, Comparison