Anti rpa32

WT U2OS-DR and HELQ^−/− U2OS-DR cells were seeded onto four-chamber tissue culture slides (Millipore) and treated the next day with 4 µM camptothecin or 10 Gy irradiation. For siRNA knockdown, cells were transfected with non-targeting or HELQ siRNA, incubated overnight and seeded onto chamber slides the next day. Cells were treated with the designated damaging agent 48 h after siRNA transfection. At the designated time points, cells were fixed, blocked and permeabilized. Cells were stained with the following antibodies: anti-phosphorylated-histone H2A.X (Ser139) (05-636, Millipore Sigma-Aldrich), anti-RAD51 (PC130 Millipore Sigma-Aldrich), anti-RPA32 (2208, Cell Signaling Technology), anti-phosphorylated-RPA32 (S4/S8) (ab87277, Abcam). Secondary antibodies were as follows: AlexaFluor 488-labelled goat anti-rabbit IgG, AlexaFluor 568-labelled donkey anti-mouse IgG, AlexaFluor 568-labelled goat anti-rat (Invitrogen). Images were obtained using a Zeiss LSM 880 confocal laser scanning microscope and analysed using ImageJ. At least 100 nuclei were counted per experiment and nuclei with >5 foci were scored as positive. Data represent the mean ± s.e.m. of at least three independent experiments, and statistical analysis was performed using two-tailed paired t-tests.

Abl pre-B cells were arrested in G₀ using 3 μM imatinib (Selleck Chemicals, #S2475) for 48 hr. MCF10A cells were arrested in G₀ by withdrawing EGF for 48 hr. To arrest cells in G₂, abl pre-B cells were treated with 10 μM RO-3306 (Selleck Chemicals, #S7747) overnight. For experiments analyzing DNA-PKcs and ATM inhibition, 10 μM NU7441 (Selleck Chemicals, #S2638) or 15 μM KU-55933 (Selleck Chemicals, #S1092) was added 1 hr prior to irradiation. After irradiation with 20 Gray, cells were allowed to recover for 3 hr. Cells were then pre-extracted with 0.05% Triton-X 100 (imatinib-treated abl pre-B cells), 0.2% Triton-X 100 (proliferating abl pre-B cells), or 0.5% Triton-X 100 (MCF10A cells) in PBS and fixed with BD Cytofix/Cytoperm solution (BD Biosciences, #554722) containing 4.2% formaldehyde. Fixed cells were stained with anti-RPA32 (Cell Signaling Technology, #2,208 S) for 2 hr at room temperature, and then treated with a fluorescent conjugated secondary antibody (BioLegend, #405,416 or BioLegend, #405418) for 1 hr at room temperature. 7-AAD was added to each sample to stain for DNA content. Cells were analyzed using a BD LSRII Flow Cytometer or a BD FACSCelesta and flow cytometry results were further analyzed using FlowJo.