C2 10

For immunoblotting, the following antibodies and dilutions were used: mouse anti-BRCA2 (1:1000, OP95, Ab-1, Merck Millipore), mouse anti-PARP-1 (1:5000, C2-10, Enzo Lifesciences), mouse anti-HSP90 (1:1000, AC88, Abcam) and HRP-conjugated Sheep anti-mouse IgG (H+L) (1:2000, Jackson ImmunoResearch). For immunofluorescence rabbit anti-RAD51 (1:10000, [43 (link)]) and an Alexa Fluor® 594 goat anti-mouse (1:1000) were used.

Cells were scraped and lysed in Laemmli sample buffer (2% SDS, 10% Glycerol and 60mM Tris pH 6.8), heated to 95 °C for 5 min and protein concentration was measured using the Lowry protein assay [35 (link)]. Size separation of proteins was achieved on a 3–8% Tris-Acetate gel (Novex, Thermo Fisher Scientific) and proteins were transferred to a PVDF membrane by wet blotting at 300 mA for two hours at 4 °C in transfer buffer (0.4 M Glycine, 5 mM Tris, 20% Methanol). Next, the membrane was blocked in 3% dry skimmed milk in PBS with 0.05% Tween-20 for at least one hour. Primary antibodies against BRCA2 (1:1000, OP95, Calbiochem, San Diego, CA, USA), BRCA1 (1:500, OP92, Merck Millipore), PARP1 (1:5,000, C2-10, Enzo Life Sciences, Farmingdale, NY, USA) and α-tubulin (1:10,000, T5168, Sigma-Aldrich, Saint Louis, MO, USA) were diluted in blocking buffer and incubated overnight at 4 °C. The secondary antibody, HRP-conjugated sheep anti-mouse IgG (1:2000, Jackson ImmunoResearch, Suffolk, UK), was incubated for 2 h at room temperature. Then, ECL substrate was added and the signal was detected on the Alliance 4.7 (Uvitec Cambridge Cleaver Scientific, Warwickshire, UK). The bands were quantified using the ‘Analyze Gels’ function in Image J (version 1.50i) and equal protein loading was verified by the α-tubulin loading control.