Analysis of ERK-dependent gene expression in immature MDDCs. (A) Effect of U0126 (UO) or PD98059 (PD) treatment on ERK1/2 phosphorylation in immature DCs after the indicated time of treatment, as determined by western blot. The levels of total ERK1/2 and GAPDH protein were determined in parallel. The histogram illustrates the ratio p-ERK/ERK for each sample, as determined by densitometric analysis of the western blot. One of 3 representative experiments is presented. (B) Relative expression of the indicated genes in immature MDDCs treated with DMSO (-), U0126 (UO), or PD98059 (PD) after the indicated time of treatment. Results are expressed as relative expression, which indicates the expression of each gene in each sample relative to its expression in DMSO-treated immature DCs at every time point (*P < .05). Shown are the means and standard deviations (SDs) of three experiments for UO and two experiments for PD-treated samples. (C) Relative expression of the indicated genes in immature MDDCs nucleofected with either an empty vector (-) or expression vector for a constitutively active (E) or a dominant negative (A) forms of MEK, as determined by real time quantitative reverse transcriptase polymerase chain reaction 24 hours after nucleofection. Results are expressed as relative expression, which indicates the expression of each gene relative to its expression in immature DCs nucleofected with a constitutively active (E) form of MEK. The graph represents the mean and standard deviation of two different experiments. The top panels illustrate the level of ERK phosphorylation (left) in one representative experiment, and the ratio p-ERK/ERK after densitometric analysis of the western blot (right) in each sample.