Figure 6

Ligand-independent activation of MET by the Biotin-conjugated anti-MET antibodies promotes degradation of the MET protein. (A) Serum-starved U373-MG cells were treated either with HGF (50 ng/ml) or PBS (control), alternatively, with the biotin-conjugated anti-MET antibodies or control IgG (each at 2 μg/ml) for various time points as indicated. The cells were directly lysed in SDS buffer and the protein levels of Y1234/Y1235-phosphorylated MET (p-MET, Y1234/5) and total MET proteins were detected by Western blotting with respective antibodies. Actin is used as the loading control. Shown is a representative set of blots from three independent repeats. (B) A quantification of p-MET protein levels in cells treated with HGF or with anti-MET antibodies using data from panel A. The relative p-MET protein levels were first normalized to the actin as loading control, and then calculated using the p-MET levels at 15 min after HGF treatment as the reference point (1.0). Data of quantitation are shown, with the mean and standard deviation (S.D.) for error bars derived from three independent repeats. (C) A quantification of total MET protein levels in cells treated with HGF or anti-MET antibodies using data from panel A. The relative total MET protein levels were first normalized to the actin as loading control, and calculated using the MET protein levels at the 0 min time points under each condition as the reference point (1.0). Data of quantitation are shown, with the mean and standard deviation (S.D.) for error bars derived from three independent repeats. (D) Serum-starved U373-MG cells were treated either with HGF (50 ng/ml) or the biotin-conjugated anti-MET antibodies (2 μg/ml), or with corresponding PBS and the normal IgG controls, for extended time points as indicated. The protein levels of total MET proteins were detected by Western blotting with respective antibodies as described in panel A. Relative levels of total MET proteins during each treatment were quantified as in panel B. Shown is a representative set of blots from three independent repeats. (E) Serum-starved U373-MG cells were pulse-treated (7 minutes treatment) with either HGF (50 ng/ml), the biotin-conjugated anti-MET antibodies (2 μg/ml) or PBS (as control) as indicated. The cells were washed and then incubated in the starvation media to recover for the indicated times before harvesting. 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 at various time points were quantified as in panel B. Shown is a representative set of blots from three independent repeats. (F) Serum-starved U373-MG cells were pulse-treated for 7 minutes with either HGF (50 ng/ml), the biotin-conjugated anti-MET antibodies (2 μg/ml), or left untreated (control). The cells were washed extensively with warm PBS to remove the unbound HGF or the antibodies, and then incubated in the starvation media for recovery for the indicated times. Cells were then fixed and immunostained with anti-MET antibodies. Arrowheads indicate the presence of MET protein on the plasma membrane. Scale bars, 20um.