Rad51

The immunofluorescence staining was performed as described previously[45 (link)]. Briefly, cells were plated onto poly-L-lysine treated glass coverslips and cultured for 24 hours in RPMI1640 medium with 10% FBS and Penicillin-Streptomycin to allow cells to adhere to the substrate. Cells were radiated with 2 Gy in a radiator before the cultural medium were changed to RPMI1640 with either DMSO or YM155 (0 hour). At 0, 2, 4, 12 hours, coverslips were rinsed twice with PBS for 5 min. Cells were fixed in fixative solution, permeabilized with 0.1% triton in PBS for 10 min at room temperature, and blocked with 3% ovalbumin in PBS (blocking solution). Primary antibodies (γH2AX from Cell Signaling Technology) and RAD51 (Novus Biologicals) were diluted at 1:200 (or as recommended by the manufacturer) in blocking solution and incubated overnight at 4°C. Cell were rinse 3 times with PBS for 5 min, incubated for 30 minutes at 37°C with the respective secondary antibodies diluted 1:200 in blocking buffer. Confocal images were obtained using an Axiovert 200 M Zeiss microscope equipped with Zen 2009 software (Carl Zeiss Microscopy, Gottingen, Germany). Number of foci per cell were counted using Image J (NIH) software. Data analysis and statistical differences were determined with Student’s t-test using the Excel software.

p16 (#550834; BDPharmingen), phosphorylated CHK1 (pCHK1) (#12302; CST), phosphorylated CHK2 (pCHK2) (#2661; CST), FANCD2 (#100182; Novus), phosphorylated H2Ax (γH2AX) (#05636 Millipore), phosphorylated SMC1 (pSMC1) (#4805S; CST), BRCA1 (#OP92; Millipore), RAD51 (#NB100148; Novus), APOBEC3A (#HPA043237; Sigma), APOBEC3B (#NBP256411; Novus), APOBEC3B (mAb 5210-87-13) (Gift from Reuben Harris lab)) [31 (link)]