Induction of c- fos in WT ES cells. A – Cells were serum-starved for 36 h and incubated with 25 μM PD98059 during the last hour of starvation where indicated. Cells were stimulated with 15% FCS or 20 μM LPA for 30 min, followed by quantitative RT-PCR. Based on relative mRNA levels, fold inductions of c-fos were calculated from three independent experiments (FCS: 82.1 ± 27.6, FCS + PD: 43.8 ± 25.7, LPA: 63.1 ± 43.5, LPA + PD: 39.7 ± 25.2). To eliminate experiment-to-experiment variations in the induction levels and to more clearly show the effect of PD98059, induction without inhibitor was set to 100% for each individual experiment and percent c-fos induction was calculated for the samples treated with inhibitor. B – Cells were serum-starved and treated as in A. Stimulation was done for 10 min and ERK1/ERK2 phosphorylation was analysed as in Figure 1. C – Cells were serum-starved and treated as in A, except that PD98059 was replaced by 5 μM Ro318220. Fold inductions from three independent experiments were FCS: 76.2 ± 42.0, FCS + Ro: 15.7 ± 10.5, LPA: 67.9 ± 20.3, LPA + Ro: 2.8 ± 1.9. Representation of the data as in A.