γh2ax antibody

Cells were grown on glass coverslips. After at least 24 h of culture, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and stained with primary antibodies over-night at 4 °C. 53BP1 (NB100-304) and FANCD2 (NB100-182) antibodies were purchased from Novus Biologicals, γH2AX antibody from Merck/Millipore (05–636), and RAD51 antibody from Santa Cruz biotechnology (sc-8349). Cells were washed three times with PBS Tween 0.1% and incubated with the secondary antibodies for 1 h (Rhodamine Red X (R6394) and AlexaFluor 488 Goat anti-mouse (A11017) or anti-rabbit (A11070), purchased from Invitrogen). DNA was stained with 4.6-diamino-2-phenyl indole (DAPI). Cells were counted positive for foci formation when >10 foci/nuclei were detected.

Cells were incubated on ice for 30 min in lysis buffer (50 mM Tris-HCl pH7.5, 500 mM NaCl and 0.5% NP40) containing the Halt^TM Protease & Phosphatase inhibitor cocktail (Thermo Scientific) and sonicated on a VibraCell 72434 (Bioblock Scientific). Cell lysates were centrifugated and the supernatant containing total soluble proteins was kept. Chromatin and soluble fractions were then prepared as previously described59 (link). Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham). Membranes were incubated with the primary antibody for a period of 1–16 hours. H2AX antibody was purchased from Epitomics (3522-1), γH2AX antibody from Merck/Millipore (05–636), ATR (SAB4200348), Lamin A/C (SAB4200236), β-actin (sc-47778) antibodies from Santa Cruz, XRCC1 (X0629), XRCC4 (HPA006801), XPA (SAB1406599-50UG) antibodies from SIGMA, FANCD2 (NB100-182) antibody from Novus Biologicals, and GAPDH (GTX100118) antibody from GeneTex. Secondary fluorescent antibodies (CF^TM770 Goat anti-rabbit (20078) or anti-mouse (20077) and CF^TM680 anti-rabbit (20067) or anti-mouse (20065), purchased from BIOTUM, were visualized on the membrane using an Odyssey Infrared Imaging Scanner (Li-Cor ScienceTec). Lamin, β-actin or GAPDH were used as internal control to normalize the protein level.

MEFs at passage four were seeded in a 96 well optical plate at a density of 10.000 cells/well. After 24 hours, MEFs were incubated with hydroxyurea (Sigma-Aldrich H8627) or UCN-01 (Sigma-Aldrich U6508) at indicated concentrations for four hours, followed by fixation with 4% paraformaldehyde (PFA). For immunofluorescence experiments, cells were permeabilized with Triton X-100 (Sigma-Aldrich T8787), blocked in IF blocking buffer (3% BSA, 0.1% Tween in PBS), and incubated with γH2AX antibody (Merck Millipore 05-636) at 4°C overnight. Samples were incubated with goat anti-mouse IgG secondary antibody, Alexa Fluor 488 (Sigma-Aldrich A-11001) for 1 hour at room temperature, and nuclei were stained with DAPI. High-content microscopy was performed using an automated Olympus IX83 ScanR microscope.

Cells were seeded equally in Petri dishes, incubated for 24 h and then treated with DMSO or SFN for 24 h. After medium change of all probes some dishes were exposed to RT. Cells were then detached from the dishes 1 h and 12 h after medium change using EDTA and resuspended in fresh medium, centrifuged, washed with PBS and finally fixated with ethanol. For quantification of γH2AX cells were again washed with PBS to remove ethanol, then permeabilized with Triton-X and incubated with γH2AX antibody (Merck KGaA, Darmstadt, Germany). Data analysis was performed with laser scanned flow cytometry as described previously [20 (link)].

The cells covered 80–90% of the glass coverslips. The cells were washed three times with PBS and fixed in 4% paraformaldehyde (Boster, Wuhan, China). Then, the cells were permeabilized with 0.1% Triton X-100 and blocked in 4% bovine serum albumin. Images were obtained using a DM-RXA2 fluorescence microscope (Leica) after incubation with γH2AX antibody (Merck Millipore, Darmstadt, Germany), a fluorescence-conjugated secondary antibody, and DAPI (4′,6-diamidino-2-phenylindole) stain (Helixgen, Guangzhou, China).