Rad51 h 92

Specific recruitment of XPA, XPC and RAD51 to the triplex site was evaluated using ChIP assays as previously described (26 (link)). Wild-type MEFs were transfected with AG30 or MIX30, and cells were collected 4 h and 6 h post-transfection. Chromatin immunoprecipitation (ChIP) was performed using the following antibodies: RAD51 (H-92, Santa Cruz), XPC (H-300, Santa Cruz), IgG (Jackson Immunoresearch Lab) and XPA (14 (link)). Samples were subjected to polymerase chain reaction (PCR) using the Advantage 2 PCR kit (Clontech) and amplified in an Eppendorf thermal cycler with the following settings: initial denaturation for 5 min at 95°C and 40 cycles at 95°C for 1 min, 1 min at 55°C and 1 min at 72°C. The primers utilized in these studies were: Primer J1, 5′- ACC TTC GAA GTC GAT GAC GGC AG and Primer J2, 5′- AGC GGA TAA CAA TTT CAC ACA GG. The PCR products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining using a BIORAD Chemidoc imaging system. The band intensity of the PCR products was quantified using Image Lab (BIORAD). The relative enrichment of the PCR products was determined via normalization against input followed by normalization to the untreated samples.

To analyze RAD51 foci formation, cells were plated on coverslips (Fisher Scientific) in a 6-well plate at a density of 150,000 cells per well and were incubated overnight in 5% FBS media. Cells were irradiated with 5 Gy then incubated at 37 °C for 6 h before being processed for RAD51 foci formation or H2AX staining as previously described [22 (link)]. Antibodies used for overnight immunostaining at 4 °C were RAD51 (H-92) (Santa Cruz) at 1/100 dilution or phosphorylated γH2AX (JBW301) (EMD Biosciences) at 1/200 dilution. Z-stack images were acquired using NIS elements software on a Nikon A1R confocal microscope with a Nikon 60× oil objective NA 1.4.