Fig. 4. Identification and characterization of p145^met, which is predominantly tyrosine phosphorylated in serum-starved 5637 cells. (A) Whole cell extracts were prepared from normally grown (control), HGF-treated (+ HGF, 125 ng/ml, 30 minutes), or serum-starved (– FCS, 24 hours) 5637 cells and immunoprecipitated (300 µg/lane) with either anti-Met antibody (IP: Met) or anti-phosphotyrosine antibody PY99 (IP: PY99). The immunoprecipitates were analyzed by immunoblotting with either anti-Met antibody (IB: Met) or anti-phosphotyrosine antibody PY99. The whole cell extracts (W, 30 µg/lane) were also analyzed by direct immunoblotting to serve as positive controls. Note that the position of p145^met matches exactly the position of the tyrosine-phosphorylated p145. Asterisks indicate the positions of p180^met, the precursor form of p145^met. (B) Whole cell extracts prepared as in A (control, + HGF, and – FCS: each 600 µg/analysis) were immunoprecipitated with anti-Met antibody and analyzed by immunoblotting with anti-Met antibody or phospho-specific anti-Met antibodies: pY1003, pY1234/1235, pY1349 or pY1365. The positions of the unphosphorylated or phosphorylated p145 [p(p)145^met] are indicated. (C) Carcinoma 5637 cells were cultured in normal (10% FCS, 1 hour) or serum-free condition (24 hours) in the absence or the presence of HGF (50 µg/ml), K252a (10 µM) and U0126 (10 µM). After treatment, cells were extracted and examined for phosphorylation of p145^met (IB: PY99) and p42/p44 MAPK (IB: pMAPK) (30 µg/lane). (D) Carcinoma 5637 cells were treated with either HGF (250 ng/ml) or serum-free medium (–FCS) for the indicated times. Whole cell extracts (30 µg/lane) were prepared and analyzed by immunoblotting with anti-Met antibody. The positions of p180^met (asterisk) and p145^met are indicated.