PGE2 binding to EP4 triggers the activation of MAPKerk1/2 and Egr-1 and mPGES-1 expression. [A] RAW264.7 macrophages (12-well plate; 5 × 105/well) were cultured in DMEM-0.1% LE-BSA for 24 h. Cells were then incubated with 10 μM butaprost (EP2 agonist), PGE2 or PGE1-OH (EP4 agonist) for 0–25 min, and lysates were prepared. Levels of phosphorylated and total MAPKerk1/2 were determined utilizing Western blot. Data are representative blots and the mean levels (± SD) of phosphorylated MAPKerk1/2 (P~erk1/2), presented as RDUs, of 3 experiments. [B,C] Macrophages (12-well plate; 5–7 × 105/well) were pre-incubated 30 min in DMEM-0.1% LE-BSA or media containing EP4 antagonists AH23848 or ONO-AE2-208, followed by PGE2. Total RNA was collected at 30 min and 18 h, and mRNA levels for Egr-1, mPGES-1 and actin were determined utilizing PCR. Data are representative blots and the mean levels (± SD) of target mRNAs, presented as RDUs, of 3 experiments.