4.7. Western Blot Analysis

Cells were lysed in lysis buffer (Sodium Sodecyl Sulfate (SDS) 1%, Tris pH 7.4 10 mM, Sodium orthovanadate 1 mM). Protein concentration was determined using the BCA Protein Assay Kit (Sigma-Aldrich, St Quentin-Fallavier, France). Samples in Laemmli buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue) containing equal amounts of total protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to PVDF (PolyVinyliDene Fluoride) membranes (Thermo Scientific, Illkirch, France), these were probed 12 h with primary antibodies PARP and β-tubulin (Cell signaling, Leiden, The Netherlands). Membranes were finally probed with secondary fluorescent antibodies (Li-COR IRDye). Antibody binding was visualized with the LI-COR odyssey Fc system (Cambridge, UK).