Quantity one 1 image analysis software

Cells were collected, washed with cold phosphate-buffered saline (PBS), and lysed in RIPA buffer. Protein concentrations were determined with a microplate reader (Bio-Rad Laboratories) and absorbance measurements were taken at 562 nm. Protein lysates were separated using SDS-PAGE (using 8%–12% SDS gels) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% nonfat dry milk in Tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature . The membranes were then incubated overnight at 4°C with primary antibodies purchased from Cell Signaling Technology (Danvers, MA, USA) that recognize: phospho-EGFR (#3777, 1:1000), EGFR (#4267, 1:1000), phospho-AKT (#9271, 1:1000), AKT (#4691, 1:1000), caspase-3 (#9665, 1:1000), PARP (#9542, 1:1000), caspase-8 (#9746, 1:1000), caspase-9 (#9508, 1:1000), DNA-PKcs (#12311, 1:1000), Rad50 (#3427, 1:1000), Ku70 (#4588, 1:1000), γ-H2AX (#9718, 1:1000), and β-actin (#3700, 1:1000). The next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit/mouse secondary antibodies (1:2000; Cell Signal Technology) as appropriate. After 1 h at room temperature, band intensities were quantified and analyzed with Quantity One 1 Image Analysis Software (Bio-Rad).