Figure 2.

EGCG inhibits Fyn kinase activity and phosphorylation of Fyn in a dose-dependent manner. (A) For the in vitro kinase assay, phosphorylation of a Fyn substrate peptide was determined with active Fyn (10, 50, or 100 ng alone or with (B) EGCG (1, 5, 10, 20 µM). (C) For the immunoprecipitation kinase assay, cells were treated or not treated with EGCG (5, 10, or 20 µM) 1 h and then treated with EGF (10 ng/mL) and harvested after 30 min. The average ³²P count was determined from three separate experiments. The asterisk (*) indicates a significant EGCG-induced change in kinase activity compared to respective control (**, P<0.01 and *, P<0.05). (D) Cells were treated with various concentrations (0, 5, 10, or 20 µM) of EGCG followed by EGF (10 ng/mL). Phosphorylation of Fyn (Thr12) (upper panel) and total Fyn protein level (lower panel) were then determined by Western blot analysis as described in Materials and Methods with specific antibodies against phosphorylation of Fyn (Thr12) or total Fyn protein.