Fig. 3. Characterization of EGF-induced Erk-1/2 activation and LTD₄-induced EGF receptor phosphorylation in Int 407 cells. The cells were pre-incubated in the absence or presence of genistein (Gen; 50 µg/ml for 30 minutes), PP1 (10 µM for 15 minutes), PTX (500 ng/ml for 2 hours), PD98059 (50 µM for 30 minutes), GF109203X (GFX; 30 µM for 30 minutes) or TPA (1 µM for 24 hours, i.e. PKC depletion). Then the cells were stimulated with 100 ng/ml EGF (5 minutes), lysed, and the lysates were separated by SDS-PAGE as described in the Materials and Methods. (A) shows a representative immunoblot with a specific anti-phospho-Erk-1/2 antibody that was then reprobed with an anti-total-Erk-1/2 antibody. (B) illustrates results from cells that were stimulated with either 80 nM LTD₄ for 3 minutes, 100 ng/ml EGF for 5 minutes or not stimulated at all (control). Lysates of these cells were separated by SDS-PAGE, immunoblotted with an anti-phospho-EGF receptor antibody and then reprobed with an anti-total-EGF receptor antibody. (C) illustrates results from cells pre-incubated with or without 2 µM PD153035 for 30 minutes and thereafter stimulated as above. Lysates of these cells were separated by SDS-PAGE, immunoblotted with a specific anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody. The blots shown are representative of five separate experiments.