Measurement of PGE2 and cAMP concentrations

For PGE₂ measurements, an enzyme immunoassay was used according to the manufacturer’s protocol (Prostaglandin E₂ Express EIA Kit—Monoclonal; Cayman Chemical Company). PGE₂ concentrations were determined in diluted basolateral cell culture medium after incubating mCCD_cl1 cells with arachidonic acid, COX inhibitors, or vehicle at 37°C for 10 min.

For cAMP measurements, mCCD_cl1 cells were lysed to release intracellular cAMP, which was quantified using an enzyme immunoassay according to the manufacturer’s instructions (cAMP Biotrak competitive enzyme immunoassay system; GE Healthcare). Cytosolic cAMP concentrations were determined in cells exposed to PGE₂ or vehicle at 37°C for 10 min. Protein concentrations were determined with a bicinchoninic acid assay (BCA Protein Assay Kit; Thermo Scientific).

All samples were measured as duplicates.