EGF activates ERK phosphorylation in LNCaP cells. LNCaP cells were preincubated with or without MEK1/2 inhibitor, U0126 (10 μ M) for 3 h followed by treatment with LY, EGF and serum as in Figures 1 and 2. (a and b) cells were harvested at 1 h post-treatment, analyzed by SDS–PAGE and subsequently immunoblotted with antiphospho ERK1/2 and anti-ERK1/2 antibodies. (c) Densitometry analysis of results from (a and b). Results are shown as the ratio of phosphorylated ERK over total ERK converted to % of the ratio obtained in cells incubated with serum (% of relative intensity). At 6, 12 and 24 h post-treatment, cells were harvested and analysed for (d) % of cells in sub-G1 and (e) caspase 3 activity. Results shown in (a and b) are from one representative experiment of at least three. (d and e) are a mean of three experiments performed in duplicates±s.e.