Phospho s139 h2ax

Immunofluorescence analyses were performed as previously described^{32 (link)}. Briefly, cells grown on coverslips were washed once with cold PBS and fixed with 4% para-formaldehyde/PBS for 10 min. Fixed cells were permeabilized with 0.2% Triton X-100/PBS on ice for 5 min. The cells were incubated overnight with primary antibodies: phospho-S139-H2AX (Millipore, 23464), RAD51 (Calbiochem, PC130), RPA (Santa Cruz Biotechnology, sc-53496), CHK1 (Abcam, ab22610) or anti-CENPF (Lifespan Biosciences, LS-B276 & LS-B3046). After being washed three times with cold PBS, the cells were incubated for 1 h with secondary anti-mouse Alexa Fluor594 (Invitrogen) at a dilution of 1:500 or anti-rabbit Alexa Fluor488 (Invitrogen) at a dilution of 1:600. The nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, 10 ng/ml). Slides were mounted in Vectashield mounting medium (Vector Laboratories). Immunofluorescence was observed with a Zeiss AxioObserver.Z1 microscope (objectives: ECPlnN 40×/0.75 DICII, resolution 0.44 μm; Pln Apo 63×/1.4 Oil DICII, resolution 0.24 μm; EC PlnN 100×/1.3 Oil DICII, resolution 0.26 μm and filters: Zeiss 43, Zeiss 38, Zeiss 49). Semi-confocal images were obtained using the Zeiss Apotome, Zeiss AxioCamMRm and Zeiss AxioVision Software. Analysis of foci intensities was performed using ImageJ software.

Antibodies: anti-DNA-RNA hybrid S9.6 (Millipore, MABE1095), HA (Santa Cruz, sc-805), Flag (Sigma-Aldrich, F3165), β-tubulin (Sigma-Aldrich, T4026), β-Actin (Santa Cruz, sc-8432), GAPDH (GeneTex, GTX100118), Chk1 (Santa Cruz, sc-8408), phosphoS345-Chk1 (cell signaling, #2341), Chk2 (Millipore, 05-649), phosphoT68-Chk2 (cell signaling, #2661), phosphoS1981-ATM (Gene Tex, GTX61739), phospho S139-H2AX (Millipore, 05-636), phospho S139-H2AX (Abcam, ab2893), H2AX (Gene Tex, GTX127340), XPF (Santa Cruz, sc-398032), XPG (Proteintech, 11331-1-AP), 53BP1 (Millipore, MAB3802), H3S10p (Millipore, 06-570), p53 (Calbiochem, OP43), p21 (Santa Cruz, sc-817), and Pol II (N-20) (Santa Cruz, sc-899). Chemicals for cell treatment: Nocodazole (Sigma, M1404), NU7741 (Santa Cruz, sc-208107), and AZD2281 (Selleckchem, S1060).