The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells. After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho VEGFR2Y996, phospho VEGFR2Y1056, SrcY461, phospho FAKY861 and phospho FAKY925. Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. (A) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. (B) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.