Rabbit anti rad51

To analyze if cells are able to use homologous recombination (HR) in an efficient manner we seeded cells onto glass slides until a confluence of 80 % was reached [19 (link),20 ]. Cells were than treated with 5 µM DNA-PK inhibitor CC-115 (Selleckchem, Houston, TX, USA) for 24 h to block the non-homologous end-joining pathway and force the cells into HR. After incubation glass slides were irradiated with a dose of 10 Gy by an ISOVOLT Titan X-ray generator and 4 h after irradiation cells were fixed with formaldehyde (4 % v/v) and blocked with a 1 % BSA containing solution. Foci of γH2AX and RAD51 were stained with primary mouse antibody anti-γ H2AX (1:1500, Merck, Darmstadt, Germany) and rabbit anti-RAD51 (1:250, abcam, Cambridge, UK). For detection secondary antibodies AlexaFluor488 goat anti-mouse and AlexaFluor594 chicken anti-rabbit (Invitrogen, Eugene, OR, USA) were used. DAPI was applied for DNA staining (10236276001, Sigma Aldrich, St. Louis, MO, USA). Images were taken by a Zeiss Imager Z2 fluorescence microscope (Zeiss, Oberkochen, Germany) and number of foci was automatically counted by Biomas software (Version V3.07/2012, MSAB, Erlangen, Germany) in minimum of 300 cells per condition.

Western blot was done to study protein levels in whole-cell protein samples or nuclear fractions. Protein samples were loaded onto a SDS polyacrylamide gel. Polyvinylidene difluoride membrane was used for the Western blot as described earlier (Laskar et al., 2011 (link)). The primary antibodies used were mouse anti-Act1 antibody (Abcam), rabbit anti-Rad51 (Abcam), mouse anti-Hsp82 antibody (Calbiochem), rabbit anti-Aha1 antibody (Invitrogen), mouse Anti-DDDDK tag antibody (Abcam), and rabbit anti-GFP antibody (Abcam) at 1:5000 dilutions. For subcellular fractionation, we used mouse anti-Nsp1 antibody (Abcam) as loading control at 1:5000 dilution. For secondary antibodies, horseradish peroxide–conjugated anti-rabbit antibody (Promega) and anti-mouse antibody (Promega) were used at 1:10,000 dilution. The Western blots were developed using chemiluminescent detection system (Thermo Fisher Scientific). Every experiment was repeated at least three times and band intensities were quantified by using ImageJ software. Mean relative densities were plotted using GraphPad Prism.