A301 844a t

Proteins were resolved by Mini Gel SDS-PAGE (Biorad^® system) and transferred to PVDF membrane (Immobilon-P^®- Millipore^®) according to standard procedures. Blocking and antibody incubations were performed in TBS - 0.2% Tween-20^® 5% milk (Marvel^®). The following primary antibodies were used: anti-Ku80, (1:1000)^{38 (link)}, anti-actin (1:5000, Santa-Cruz^® sc-1615), anti-H2AX-P (1:1000, Abcam® ab11174), anti-H3 (1:2000, Abcam^® ab12079), anti-flag (1:2000, SIGMA^® F1804 and F2425), anti-mono-ADPr AbD33205 (1:1000)^{34 (link)}, Anti-H3S10-p (1:1000, Bethyl^® A301-844A-T, and Abcam^® ab5176). Global ADP-ribosylation sigma was detected using the reagent anti-PAN-ADPr (1:2000, Merk^® MABE1016). Appropriate HRP-conjugated secondary antibodies were used anti-mouse (1;10000, DAKO^®), anti-rabbit (1;10000, DAKO^®), anti-goat (1;10000, DAKO^®) and anti-human (1:5000)^{34 (link)}. Immuno-reactive bands were detected by chemo-luminescence induced by Immobilon^® western substrate (Millipore^®), detected with the LI-COR^® Odyssey-Fc machine and quantified using Image-Studio^® software.

Cells were plated onto glass coverslips and allowed to attach. Cells were fixed with 4% paraformaldehyde (AlfaAesar^®) for 15 min. Where necessary, prior to fixation, soluble protein was pre-extracted using 0.5% Triton X-100 in KK2 for 5 min at 4 °C. Cells were then permeabilised in 0.5% Triton X-100 in KK2 for 10 min, and blocked in 1% bovine serum albumin (Sigma^®) for 1 h. Coverslips were stained with primary antibody (2 h, room temperature) against H3S10-P (1:500, Bethyl^® A301-844A-T) or γH2AX (1/500, Abcam^® ab11174), washed extensively in KK2-0.01% Tween-20^®, and stained with fluorescently labelled secondary antibody (2 h, room temperature) anti-mouse FITC (1:500, DAKO^®, F0232) or anti-rabbit TRITC (1:500, DAKO^®, R0156). Following further washing, cells were mounted onto slides in Vectashield^® containing DAPI (Vector Laboratories). Samples were visualised using a microscope Zeiss IX71 equipped with a 10X dry objective and a 100X oil immersion objective lens and a Hamamatsu^® Orca-R² camera. Pictures were analysed with ImageJ^® software.