Prolonged, not immediate TGFα stimulation promotes Met phosphorylation. OSA8 cells were serum starved for 2 h and then left unstimulated, or stimulated with HGF (50 ng mL–1), TGFα (100 ng mL–1) or a combination of both for 15 min. (A) Western blot analysis was performed on canine OSA8 cell lysates for EGFR phosphorylation. HGF stimulation did not promote EGFR phosphorylation. (B) Met was immunoprecipitated from canine OSA8 cells followed by western blot analysis. TGFα stimulation did not promote Met receptor phosphorylation. Thus, although a co-association was initially seen with Met and EGFR, a reciprocal activation of the receptors did not occur with an immediate HGF or TGFα stimulation. (C) For the prolonged TGFα stimulation, OSA8 cells were serum starved for 2 h before treatment with TGFα (100 ng mL–1) for a total of 12, 24 and 36 h followed by immunoprecipitation of Met. Western blot analysis was performed and showed phosphorylation of Met occurred with 12, 24 and 48 h of exposure to TGFα. Controls (C) included serum starved nonstimulated and 15-min HGF (50 ng mL–1) stimulated OSA8 cells. TGFα promoted Met phosphorylation that was evident at 12, 24 and 48 h.