Figure 4
Pre-treatment of the serum-starved cells with the biotin-conjugated anti-MET antibodies renders the cells refractory to the subsequent HGF stimulation. (A) U373-MG cells were serum-starved for 3 hours. Cells were pulse-treated (1st treatment) with PBS (phosphate buffered saline, control) or HGF (50 ng/ml) for 7 minutes, washed extensively with warm PBS buffer, and then incubated in the serum-starvation medium for 30 minutes to recover. They were then re-stimulated (2nd treatment) with HGF or PBS (as control) for another 7 minutes. Cells were fixed and immunostained with the anti-Y1234/1235-phosphorylated MET antibodies. MET receptor clusters (patches) are indicated by arrows. (B) Serum-starved U373-MG cells were pulse-treated (1st treatment) with the Biotin-conjugated anti-MET antibodies or biotin-conjugated IgG (each at 2 μg/ml) for 7 minutes, washed extensively with warm PBS buffer, and then incubated in the serum-starvation medium for 30 minutes to recover. Cells were then stimulated (2nd treatment) with HGF (50 ng/ml) or PBS (control) for another 7 minutes. Cells were fixed and immunostained with streptavidin (pre-labeled with Brilliant Violet 421, green) and anti-Y1234/1235-phosphorylated MET antibodies (secondary antibody conjugated with Alexa Fluor 647, red). MET receptor clusters (patches) are indicated by arrows. Scale bar in B, 20 μm. (C) Serum-starved U373-MG cells were treated as in (A,B), with the 1st treatment being either HGF (50 ng/ml) or PBS control (lanes 1–5) or alternatively with the biotin-conjugated anti-MET antibodies or the control IgG (each at 2 μg/ml) (lanes 6–9), for 7 minutes, as indicated. Cells were washed extensively with warm PBS and then recovered in the serum-starvation medium for 30 minutes. Cells were subsequently stimulated (2nd treatment) with HGF (50 ng/ml) or PBS (control) for another 7 minutes, as indicated. Cells were lysed and the levels of the Y1234/1235-phosphorylated MET and total MET proteins were analyzed by Western blotting with specific antibodies as indicated.
