Amersham protran premium 0.45 m nitrocellulose membranes

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.).

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, 1 mM Na3VO4) and a protease inhibitor cocktail (P8340; Sigma Aldrich), after 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h proton IR with 4 Gy. 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 ATM, ATR, Rad51, Poly(ADP-ribose) polymerase (PARP)-1, Ku70, DNA ligase IV, phospho-histone γH2AX, TRAIL-R2, and the NF-ĸB pathway components IKKα, IKKβ, phospho IKKα/β, IĸB, phospho IĸB, p50, phospho p50, p65, phospho p65 (Cell Signaling Technology, Danvers, MA) and PCNA, MSH3, EXO1, and β-actin (Abcam, Cambridge, UK) as 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) and images were processed with the ImageLab 6.0.1 Software (BioRad Laboratories Inc; www.bio-rad.com/de-at/product/image-lab-software).