Cells were collected in dPBS by scraping and lysed in
RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with
protease (Merck Sigma) and
phosphatase (Merck Sigma) inhibitors. Extracted proteins were quantified using the
Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Subsequently, 40 µg of protein was loaded on a precast gel
NuPAGE Novex 4–12% Bis-Tris (Thermo Fisher Scientific). Proteins were then transferred onto a nitrocellulose membrane using an iBlot2 dry blotting system with
iBlot2 transfer stacks (Thermo Fisher Scientific). Membranes were incubated for 1 h at room temperature in a blocking solution of 5% BSA in Tris buffer saline 0.05% Tween-20 (TBS-T). Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against γh2AX (1:2000) (Merck Millipore#05-636) and actin (1:5000) (Sigma-aldrich #A5441). The next day, membranes were incubated with
HRP-linked anti-mouse secondary antibody (Cell Signaling Technology, 7076S) used at a dilution of 1:4000 in 5% milk in TBS-T. Bound antibodies were visualized using
SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Images were acquired using the
ImageQuant LAS 4000 (GE Healthcare).
Parik S., Fernández-García J., Lodi F., De Vlaminck K., Derweduwe M., De Vleeschouwer S., Sciot R., Geens W., Weng L., Bosisio F.M., Bergers G., Duerinck J., De Smet F., Lambrechts D., Van Ginderachter J.A, & Fendt S.M. (2022). GBM tumors are heterogeneous in their fatty acid metabolism and modulating fatty acid metabolism sensitizes cancer cells derived from recurring GBM tumors to temozolomide. Frontiers in Oncology, 12, 988872.
