Rabbit anti met

To determine the protein levels, cells (1 × 10⁶) were lysed on ice with RIPA lysis buffer before radiation, or 1-h, 24-h, 48-h, and 5 days after radiation with 4 Gy. Protein concentrations were measured with BCA protein Assay Kit (Pierce Chemical Co., USA), and 20 μg from each sample was separated on an SDS-PAGE gel and transferred to a PVDF membrane by electroblotting. After blocking the membrane with 5% nonfat dry milk in TBS with Tween, it was incubated with the primary antibodies: Rabbit- anti Met (1:1000; Cell Signaling, #8198), rabbit- anti phospho Met (Tyr1234/1235) (1:1000; Cell Signaling Technology, #3077), and mouse and rabbit anti- β-actin (1:1000 Abcam, ab8277 and ab6709). Subsequently, the membranes were incubated with secondary goat-anti-mouse and goat-anti-rabbit immunoglobulins (IRDye 680RD and 800 CW; Li-Cor Biosciences). Detection of bound antibodies was analyzed with an Odyssey infra-red imaging system (Li-Cor Biosciences). To evaluate the potential of c-Met to be phosphorylated in response to hepatocyte growth factor (HGF), the cells were stimulated with 50 ng/ml HGF (Thermo Fisher Scientific, Ghent, Belgium) for 10 min immediately before lysis.