Figure 7
Biotin-conjugated anti-MET antibodies promote degradation of the MET protein in a proteasome-sensitive manner. (A) Serum-starved U373-MG cells were treated either with HGF (50 ng/ml) (lanes 4–6) or the biotin-conjugated anti-MET antibodies (2 μg/ml) (lanes 10–12), or with the corresponding controls PBS (lanes 1–3) or the control IgG (2 μg/ml) (lanes 7–9), for 7 minutes as indicated. To each set of the cells, either DMSO (control), MG-132 (10 μg/ml), or bafilomycin A1 (BAF, 100 nM) were then added and incubated for further 2 hours. The cells were lysed in SDS buffer and the protein levels of total MET protein were detected by Western blotting with anti-MET antibodies. Actin is used as the loading control. Relative levels of total MET proteins during each treatment were quantified normalized to the actin load control, and then calculated using the MET protein levels in the PBS/DMSO control sample (lane 1) as the reference point of 1.0. Data of quantitation is obtained from three independent repeats, and a set of blots from one of these experiments is shown. (B) Serum-starved U373-MG cells were treated either with APC-conjugated anti-MET antibodies (1 μg/ml, lanes 4–6) or control IgG (1 μg/ml, lanes 1–3) for 7 minutes as indicated. Cells were then treated with DMSO, MG-132 or BAF, as in panel A, and processed similarly as in panel A. Relative levels of total MET proteins during each treatment were quantified, normalized to the actin load control, and then calculated using the MET protein levels in the IgG-APC/DMSO control sample (lane 1) as the reference point of 1.0. Data of quantitation is obtained from three independent repeats, and a set of blots from one of these experiments is shown. (C) Serum-starved LN229, LN18 and T98G cells were treated either with biotin-conjugated anti-MET antibodies (2 μg/ml) (lanes 5–7) or HGF (50 ng/ml) (lanes 1–3) or left untreated (lane 4) for 7 minutes as indicated. Cells were then treated with DMSO, MG-132 or BAF, as in panel A. The cells were lysed in SDS buffer and the protein levels of total MET protein were detected by Western blotting with anti-MET antibodies. Actin is used as the loading control.
