PGE₂ increases CREB Ser133 phosphorylation, which is involved in STAT5 transactivation.

(A) Western blot analysis of nuclear protein extracts of AS-E2 cells that were pretreated with or without PGE₂ (1 μM) and stimulated with or without EPO (2 U/mL) for the indicated periods, using antibodies against Ser133-phosphorylated (αP-S-CREB) and total CREB (αCREB). Shown is a representative blot of 3 independent experiments. (B) STAT5 transactivation assay of AS-E2 cells that had been cotransfected with control vector (pcDNA3) or expressing vector for wild-type or Ser133Ala CREB. Cells were starved for 16 hours, pretreated with or without PGE₂ (1 μM), and stimulated with or without EPO (2 U/mL) for 7 hours. Luciferase activities were corrected for β-galactosidase activities and expressed as percentage transactivation of EPO-stimulated control cells without PGE₂ pretreatment. The mean values of 3 independent experiments are presented, and SEM values are indicated by bars.