Egfr and p egfr

Cells pretreated with nimotuzumab received irradiation as described above. Cytoplasmic and nuclear extracts were prepared according to the directions for using the nuclear and cytoplasmic extraction kit (Beyotime, Nanjing, Jiangsu, People’s Republic of China) 6 hours after irradiation. Protein concentration was determined by the BCA protein assay kit (Servicebio, Wuhan, Hubei, People’s Republic of China). Protein lysates were then separated on an 8% or 12% SDS-PAGE gel depending on the protein molecular weight and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Immunoblots were blocked in 5% protease-free bovine serum albumin for 1 hour and probed with γ-H2AX (Abcam, Cambridge, UK), EGFR and p-EGFR (Cell Signaling Technology, Beverly, MA, USA), DNA-PK (Cell Signaling Technology, Beverly, MA, USA), p-DNA-PK (Epitomics, Burlingame, CA, USA), as well as GAPDH and Lamin B1 (Goodhere, Hangzhou, Zhejiang, People’s Republic of China) antibodies. The membranes were continually incubated with appropriate horseradish peroxidase secondary antibodies (Invitrogen, Carlsbad, NM, USA) after washing, and then we detected protein with a chemiluminescence kit (Invitrogen) and visualized the bands on an X-ray film. GAPDH and Lamin B1 were used as an internal control for cytoplasmic and nuclear proteins, respectively, to balance equal loading.