Rad51 14b4

Antigen retrieval was performed using citrate buffer (10 mM, pH 6) or high-pH antigen retrieval solution (Vector). Sections were blocked in 10% goat serum and incubated with antibodies overnight at 4°C followed by fluorophore-conjugated antibody for immunofluorescence or HRP-conjugated secondary antibodies (Vector) for immunohistochemistry. Antibodies used were phospho DNA-PKcs-S2056 (Abcam) and phospho-histone H2AX Ser139 (γH2AX, 20E3, Cell Signaling Technology) on human and mouse tissue; RAD51 (14B4, Genetex), T1α (clone 8.1.1, DHSB), and cleaved caspase 3 Asp175 (5A1E, Cell Signaling Technology) on mouse tissue; and T1α (NC-08, Biolegend) on human tissue. Nuclei were counterstained with DAPI where appropriate. Images were acquired using a DeltaVision fluorescence microscope (Applied Precision).

Total protein was extracted from exponentially growing cells at passage 8–10 and 40 g/mL were resolved by SDS-PAGE using a 4–15% gradient gel (Bio-Rad Laboratories). After transfer and blocking overnight at 4 °C in Odyssey Blocking Buffer (Li-Cor, Lincoln, NE, USA) proteins were detected by primary antibodies against BRCA1 [2A-9] (1:500, kindly provided by Stephen Smith, Leibnitz Institute, Jena, Germany), ATR [N-19] (Santa Cruz, St. Cruz, CA, USA, 1:1000), CHK1 [2G1D5] (Cell Signaling, Danvers, MA, USA, 1:750), RAD51 [14B4] (1:2.000, GeneTex, Irvine, CA, USA), MRE11A [12D7] (Abcam, Cambridge, UK, 1:500), pCHK1 [Ser296] (Cell Signaling, 1:1000), -actin [AC-74] (1:50.000, Sigma, St. Louis, MO, USA). Primary antibodies were detected with IRDYE 680 conjugated anti-mouse IgG, IRDYE 800 conjugated anti-rabbit IgG (Li-Cor, 1:7500), IRDYE 680 conjugated anti-rabbit IgG (Licor, 1:7.500 or 15.000) or IRDYE 800 conjugated anti-mouse IgG (Li-Cor 1:7.500 or 15.000). Quantitative and qualitative analysis was done by using Li-Cor Odyssey (Li-Cor, Lincoln, NE, USA).