Nbp2 24737

Anti-APEX1 antibody, catalogue # NB100-101 was purchased from Novus Biologicals, Inc., (Littleton, CO). Anti-APEX2 antibody is developed by us. Anti-RAD51 antibody, catalogue # SC-8349 was purchased from Santa Cruz, Dallas, TX. Antibodies against GAPDH (catalogue # mAb2118), HP1 (catalogue # 261), and γH2A.X (Ser139, catalogue #2577) were from Cell Signaling Technology, Inc., Danvers, MA. For immunoprecipitation studies, anti-APEX1 antibody (Cat. # ab194, Abcam) and anti-p73 antibody (Cat. # NBP2-24737, Novus Biologicals) were used.

Chromatin immunoprecipitations were performed using EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore). Cells were fixed in formaldehyde (1%) for 10 min at room temperature. Glycine was then added to a final concentration of 0.125 M to quench the reaction. Cells were washed twice in cold PBS and once with PBS containing protease inhibitors, incubated in lysis buffer with protease inhibitors on ice for 15 min, and centrifuged at 800 × g for 5 min at 4 °C. The supernatant was removed and the nuclear pellets re-suspended in nuclear lysis buffer with protease inhibitors and incubated on ice for 10 min before sonication. Sonication was performed using a probe sonicator in 1.5 ml tubes. Sonication conditions involved 11 cycles at 75% amplitude, 30 s pulse, 1 min on ice, followed by 1 cycle at 80% amplitude. The lysates were centrifuged at 12,000 × g for 10 min at 4 °C and the chromatin supernatants were used for immunoprecipitation, using 4 µg of APEX1 antibody (catalogue # ab194, Abcam) or p73 antibody (catalogue # NBP2-24737, Novus Biologicals) or IgG controls. Real-time PCR reactions, using primers for amplification of a region of RAD51 promoter, were conducted to assess the fraction of RAD51 promoter DNA associated with proteins under investigation. Primers specific to GAPDH promoter region were used as negative control.