Axioimager m2 microscope system

Whole-cell lysate preparation and western blotting were performed as previously described.^{22 (link), 24 (link)} For immunofluorescent staining, cells were grown on poly-D-lysine-coated culture slides (BD Pharmingen, San Diego, CA, USA), washed in phosphate-buffered saline (PBS), fixed in PBS containing 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and blocked in PBS containing 5% bovine serum albumin. The cells were incubated with indicated primary antibodies for 2 h at room temperature, washed with PBS and incubated with Alexa-568- and Alexa-488-conjugated secondary antibodies for 1 h (Invitrogen). Cells were then washed with PBS and mounted in Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Images were acquired from a Zeiss AxioImager M2 microscope system equipped with a Plan-Apochromat 63 × /NA 1.40 objective, an AxioCam MRm CCD camera and AxioVision software (Carl Zeiss, Oberkochen, Germany). Anti-Chk2 total (Cell Signaling, Beverly, MA, USA), anti-phosphorylated Chk2 at Thr68 (Cell Signaling), anti-phospho-histone H3 (EMD Millipore, Billerica, MA, USA), anti-β-actin (Sigma) and anti-Brca1 total (Santa Cruz, Dallas, TX, USA) antibodies were purchased from the indicated vendors. Antibodies against total and phosphorylated forms of DNA-PKcs were described previously.^{22 (link)}