Trail r2

Whole-cell protein extracts were prepared with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 1% NP-40, and 1 mM Na3VO4) and a protease inhibitor cocktail (P8340; Sigma Aldrich), 1 h and 24 h after 2, 4, and 8 Gy X-ray/proton/C-ions IR. Protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The proteins were separated by SDS-PAGE and were blotted on Amersham™ Protran™ Premium 0.45 µM nitrocellulose membranes (GE Healthcare Life Science, Little Chalfont, UK). Primary antibodies against the DNA damage key players phospho-p53, phospho-ATM, phospho-ATR, the death receptor TRAIL-R2, the DNA damage marker phospho-histone γH2AX (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Abcam, Cambridge, UK) as the loading control were used. Blots were developed using a horseradish peroxidase-conjugated secondary antibody (Dako) for 1 h and the Amersham™ ECL™ prime Western blotting detection reagent (GE Healthcare). Chemiluminescence signals were detected with the ChemiDocTouch Imaging System (BioRad Laboratories Inc., Hercules, CA, USA) and respective images were processed with the ImageLab 5.2 Software (BioRad Laboratories Inc.).

Antibodies were sourced as follows: AMG655 (conatumumab) was sourced from Amgen Inc (Thousand Oaks, CA, USA); FADD mouse monoclonal antibody was from BD transduction laboratories (catalog #556402); N‐terminal specific procaspase 8 antibody was purchased from Abcam (catalog #ab32125); C‐terminal specific procaspase‐8 antibody was purchased from Enzo^® life sciences (catalog # ALX‐804‐242‐C100); FLIP antibody (NF6; catalog # AG‐20B‐0056‐C100) was purchased from Adipogen; TRAIL‐R2 was purchased from Cell signalling technology (catalog #3696); and procaspase‐10 was purchased from MBL international (catalog #M059‐3). Horseradish peroxidase‐conjugated secondary antibodies were purchased from cell signalling technology; LI‐COR mouse (IRDye^® 800CW) and rabbit (IRDye^® 680CW) secondary antibodies were purchased from LI‐COR. MS‐275 (entinostat) was obtained from Selleck Chemicals (Newmarket, UK); annexin V‐FITC was obtained from BD biosciences; and z‐Val‐Ala‐Asp(OME)‐FMK (zVAD‐FMK) was purchased from Sigma‐Aldrich (Gillingham, UK). Transfections were completed using FuGENE^® HD transfection reagent (Promega, UK). The Dynabead^® antibody coupling kit and a Dynamag™‐2 magnetic rack were obtained from Life Technologies (Paisley, UK). FADD peptides were generated by Almac Sciences (Edinburgh, UK).