Relative expression of COX-2 in PA cell lines. Basal expression of COX-2 was determined by Western blot analysis in PA cell line lysates and quantified by densitometry. A: Representative Western blot. All cell lines were acquired from the American Type Culture Collection (ATCC, Manassas, VA) and propagated in ATCC recommended media. Cells were grown to 80% confluence before preparation of cell lysates. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes using standard protocols. The membranes were probed with antibodies to COX-2 (mouse monoclonal, Cayman Chemical, Ann Arbor, MI) and β-actin (rabbit monoclonal, Cell Signaling Technology, Danvers, MA) or GAPDH (mouse monoclonal, Novus Biologicals, Littleton, CO) as total protein loading controls. B: Densitometric analyses. Autoradiographs of Western blots were quantified using ImageJ software (National Institutes of Health, http://rsb.info.nih.gov/ij/). COX-2 intensity was first normalized to the corresponding loading control (β-actin or GAPDH) and then normalized to the BxPC-3 ratio for each blot. Data represents the combined results of four independent experiments (mean ± SEM).