Polyclonal anti γh2ax ps139

Following transient transfection and/or drug treatment, whole-cell extracts (WCE) were prepared and immunoblots were performed as previously described [55 (link)]. The primary antibodies used to detect proteins were monoclonal anti-BRCA1 DO-9 (1: 200, Santa Cruz), monoclonal anti-HSP60 (1:1000, Sigma), monoclonal anti-HA (1:1000, Babco), polyclonal anti-pBRCA1Ser1497 (1:50, Upstate), polyclonal anti-γH2AX[pS139] (1:200, Cell Signaling Technology), monoclonal anti-Plk1 (1:500, Zymed), monoclonal anti-Myc epitope sequence (1:1000, Calbiochem) and polyclonal anti-Kap1 (1:2000; Abcam). The secondary antibodies were peroxidase-conjugated IgG (1:5000, Cell Signaling). Immunoblot signals were detected by using ECL (Pierce). For immunoprecipitation, 500 μg to 1 mg of precleared WCE were incubated with 10 μg of rabbit polyclonal anti-BRCA1 antibody (BD Biosciences) or 2 μg of rabbit polyclonal HA-probe Y-11 (Santa Cruz) or with 5 μg of rabbit polyclonal anti-Plk1 antibody (Calbiochem) at 4°C overnight. Immune complexes were recovered with protein A-sepharose beads (Pharmacia) and washed three times using lysis buffer. Then beads were subjected to Western Blot analysis. Control immunoprecipitation was performed without antibody.

Cells were pre-extracted, fixed and permeabilized as previously described [57 (link)]. Incubation with relevant primary and secondary antibodies was carried out sequentially for 1 h each at room temperature. The primary antibodies used to detect proteins were monoclonal anti-BRCA1 DO-9 (1: 200, Santa Cruz), monoclonal anti-HA (1:1000, Babco), polyclonal Rad51 (1:200; Santa Cruz), and polyclonal anti-γH2AX[pS139] (1:100, Cell Signaling Technology). Secondary antibodies were Alexa Fluor^® 488 anti-mouse IgG (1:1000; Molecular Probes) and Alexa Fluor^® 594 anti-rabbit IgG (1:1000; Molecular Probes). DNA was stained using 0.05 μg/ml DAPI (Sigma-Aldrich) for 5 min. Slides were mounted with fluorescent mounting medium (Dako).
Images were captured using a confocal laser microscope (FV1000 Olympus; module TIRF with Hamamatsu OrcaR2 camera; Olympus Corporation) with a Plan-Apochromat 60xNA 1.40 oil immersion lens or a Plan-Apochromat 40X 0.95 lens and 405-, 488- and 559-nm lasers excitation. Images were taken with the same exposure time when comparing experimental conditions and were analyzed using Image J software (NIH).