Figure 5
Pre-treatment of the serum-starved cells with the biotin-conjugated anti-MET antibodies caused the activation of AXL refractory to the subsequent HGF stimulation. (A) Live serum-starved U373-MG cells were treated with biotin-conjugated goat anti-MET antibodies or biotin-conjugated control IgG (each at 2 μg/ml) for 7 minutes. Cells were then washed, fixed and immunostained with the streptavidin-conjugated to Brilliant Violet 421 to detect the biotin-conjugated antibodies (purple), a rabbit anti-Y779-phosphorylated AXL antibody for the activated AXL protein (secondary antibody conjugated with Alexa Fluor 647, red), and an anti-MET antibody pre-conjugated to Alexa Fluor 488 for total MET protein (green). (B) U373-MG cells were serum-starved for 3 hours. Cells were pulse-treated (1st treatment) with the biotin-conjugated anti-MET antibodies or control IgG (each at 2 μg/ml), PBS (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 (50 ng/ml) or PBS (as control) for another 7 minutes. Cells were fixed and immunostained with the anti-Y779-phosphorylated AXL antibody or with an anti-MET antibody. MET receptor clusters (patches) are indicated by arrows. Scale bars in (A,B), 20 μm. (C) Serum-starved U373-MG cells were treated as in B, with the 1st treatment being either HGF or PBS control (lanes 1–4) or alternatively with the biotin-conjugated anti-MET antibodies (2 μg/ml) (lane 7, 8) or control IgG control (2 μg/ml) (lanes 5, 6), 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, anti-Y779-phosphorylated AXL antibody, total AXL, phosphorylated AKT (p-AKT), total AKT, were analyzed by Western blotting with specific antibodies as indicated.
