Western Blot Analysis

The response of DNA damage biomarker (γH2AX) and proteins involved in repair of DNA double-strand breaks (DNA-dependent protein kinase [DNA-PK] and Rad51) and apoptosis (caspase 3 and 7 and poly-ADP ribose polymerase [PARP]) to BO-1978 was determined by Western blotting [26]. Briefly, H460 cells were treated with various concentrations of BO-1978 for 72 hours or 2 μM for different time periods. The whole cell extracts were then electrophoretically separated on an SDS polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences). After blocking, the membrane was incubated with primary antibodies and then with horseradish peroxidase–conjugated antirabbit or antimouse secondary antibodies. Western blot signals were visualized by chemiluminescence using SuperSignal West Pico chemiluminescence reagent (Pierce). Antibodies against Rad51 and β-actin were obtained from Genetex; caspases 3 and 7, PARP, DNA-PK, and secondary antibodies from Cell Signaling; and γH2AX from EMD Millipore.