Protein Extraction and Western Blot Analysis

Cells were washed with ice-cold PBS, pelleted, and lysed in 50 mM Tris–HCl at pH 8.0, 150 mM NaCl, 0.5% SDS, and 1% NP-40 supplemented with a Halt Protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and Phosphatase inhibitor cocktails 2 and 3 (Sigma, St. Louis, MO, USA) on ice for 30 min. Lysates were then cleared by centrifugation for 10 min at 14,000× g. Total protein concentration was measured using a DC protein assay (Bio-Rad, Hercules, CA, USA) and 10–50 µg of protein was mixed with loading buffer. For the analysis of H2A and γH2AX, protein samples were separated on 4–15% Mini-PROTEAN^® TGX™ Precast Protein Gels (Bio-Rad) and transferred to a PVDF membrane using Trans-Blot Turbo RTA Mini 0.2 µm PVDF Transfer Kit (Bio-Rad). The primary antibodies used were: Histone H2A Antibody II (#2578, Cell Signaling), Phospho-Histone H2A.X (Ser139) (20E3), and Rabbit mAb (#9718, Cell Signaling). HRP-conjugated anti-rabbit antibody (Sigma) was used as secondary antibody. Immuno-reactive bands were detected on the ChemiDoc MP system (Bio-Rad) using the Clarity Western ECL substrate (Bio-Rad).