Western blotting protocol

To assess the differential cytotoxicity of X-rays, we analyzed γH2AX for DSBs and Bax for apoptosis in MCF7 and MDA-MB-231 cell lines using the Western blot technique after exposure of 2 Gy, 5Gy, and 8.5Gy. After the cells were irradiated they were incubated in a CO₂ incubator for 30 min before γH2AX was analyzed because this marker reached maximum levels at 30 min after irradiation,^[17] and 4 h in a CO₂ incubator after irradiation for Bax (apoptosis regulator BAX). The Western blotting protocol was performed as follows: the cells were lysed and Bradford protein assay was used for as a protein assay. Primary antibodies γH2AX (cat no. 2577, Cell Signalling) and Bax (cat no.2772, Cell Signalling) were diluted 1:1000 in a blocking buffer and incubated at 4°C overnight in a slow shaker. The primary antibodies were then removed and the membranes were washed three times with tris-buffered saline + Tween 20 (TBST) and shaken for 10 min at room temperature in a fast shaker. A secondary antibody was added (anti-rabbit) horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Santa Cruz, TX, USA) and diluted 1:2000 in blocking buffer for 1 h in a slow shaker at room temperature. The secondary antibody was removed and washed three times with TBST for 10 min on a slow shaker at room temperature. The housekeeping gene β-actin (Human/Mouse/Rat beta – Actin Antibody, MAB8929) was used for normalization, as the same protocol as mentioned above was used for the targeted proteins γH2AX and BAX. Proteins were electro transferred to polyvinylidene difluoride membranes (Trans-Blot Turbo Transfer Pack, Bio-Rad Laboratories, Hercules, CA, USA) in a blotting machine (Bio-Rad). Immunoreactive bands were visualized using Amersham ECL Prime WB detection reagent (GE Healthcare Europe, Milan, Italy). Images were acquired using Image 6 quant LAS 500 (GE Healthcare Europe), and densitometric analysis was performed using ImageJ software (Corning).