For Western blot analysis of phospho-p70S6 kinase, p70S6 kinase, phospho-pS1981 ATM and total ATM, whole-cell extracts were prepared with RIPA buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. The Bradford method was used to estimate protein concentrations, and gels were run with 10–25 µg of protein. Immunofluorescence staining of cells was performed as previous described, with modifications (23 (link)). Antibodies were as follows: anti-phospho-γ-H2AX, anti-rad51 and anti-CtIP for primary antibodies; Rhodamine Red™-X and Alexa Fluor® 488/568 were used for secondary antibodies (Molecular Probes®, Grand Island, NY). The cells were fixed, permeabilized and blocked according to the recommended protocol from the supplier and incubated with primary antibodies for either 2 h or overnight at 4°C. Secondary antibodies were incubated for 1 h at room temperature for immunofluorescence. The slides were mounted with Vectashield® hardset mounting media containing DAPI. The images were acquired and recorded using the Zeiss Axio Imager at 63× magnification (Carl Zeiss MicroImaging Inc., Thornwood, NY).