Ab124763

T24 cells (7×10⁵ cells/ml) transfected with shNull or shRNF8 vector were plated on glass coverslips and exposed to X-ray irradiation (5 Gy) or mock-treated (0 Gy) for 1 h. Then, the cells were fixed with 4% paraformaldehyde for 15 min. After blocking with 3% bovine serum albumin/PBS containing Tween-20, the fixed cells were incubated overnight with specific antibodies and washed twice with PBS. Next, the cells were incubated with secondary antibodies for 30 min, washed twice with PBS and counterstained with 1 μg/ml DAPI (Invitrogen). The primary antibodies used were as follows: anti-γ-H2AX (Abcam, ab22551), anti-MDC1 (Abcam, ab11169), anti-53BP1 (Abcam, ab21083), anti-BRCA1 (Abcam, ab16780) and anti-RAP80 (Abcam, ab124763). Images were viewed and acquired using a 60X oil objective lens on a Zeiss fluorescence microscope controlled by AxioVision 4.8 software. All steps were performed at room temperature. At least 100 morphologically intact cells were examined.

Cells were collected and disrupted in lysis buffer (50 mM Tris pH 7.5, 20 mM NaCl, 1 mM MgCl₂, 0.1% SDS supplemented with protease and phosphatase inhibitors (Roche) and 0.1% endonuclease benzonase (Millipore)). After 10 min of incubation at room temperature, the lysates were combined with 4× Laemmli buffer containing β-2-mercaptoethanol and denatured by boiling. The proteins were separated by SDS-PAGE. The antibodies used were directed against FANCD2 (sc-20022, Santa-Cruz), actin (sc-1616, Santa-Cruz), vinculin (ab18058, Abcam), RAP80 (ab124763, Abcam), GAPDH (ab9484, Abcam), lamin B1 (ab20396, Abcam), histone H3 (ab1791, Abcam), Tip60 (sc-5725, Santa-Cruz), 53BP1 (MAB3802, Millipore), H4K16Ac (07-329, Millipore), H3K9Me3 (6F12-H4, Millipore), H3K9Ac (39585, Active Motif), H4K20Me2 (39174, Active Motif) and HA (26183, Thermo).