Western Blot Analysis of Breast Cancer Cells

As described in detail previously [44 (link)], breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer’s protocol (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 µg of protein were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes (Millipore). Membranes were subjected to immunoblotting with antibodies against TDP1 (Abcam AB4166), PARP1 (Cell Signaling Technology #9542), and tubulin (Sigma). Bound antibody was visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and chemiluminescence detection by the ChemiDoc imaging system (Bio-Rad).