Figure 5.

The EGFR kinase activity is necessary for the W-13-induced effects. COS-1 cells were preincubated with tyrphostin (1 μM; A) or 10 μg/ml Ab225 (B and C) for 20 min at 37°C. Then, incubation was followed for 60 min with W-13 and/or 25 ng/ml EGF for 10 min (A and B). An equal amount of protein was electrophoresed and phosphotyrosine of EGF-R or MEK activation was detected by Western blotting with antiphosphotyrosine RC-20 antibody HRP-conjugated or polyclonal antibody to phospho-MEK, respectively.