Fig. 2.

Western blot analysis of the over-expressed recombinant proteins in HEK293 cells. The cells were grown for 24 h on 100 mm culture dishes until they were about 95% confluent and then transfected with the purified cDNA plasmid (pcDNA3.1(+)) using Lipofectamine 2000™ following the manufacturer's instructions (Invitrogen). For the co-transfection (lane 2), the cells were transfected with 12 µg of human COX-2 cDNA plasmid and 12 µg of human mPGES-1 cDNA plasmid. For the COX-2-10aa-mPGES-1 (lane 1) and pcDNA 3.1(+) (negative control, lane 3), 24 µg of each plasmid were used for each individual transfection. Approximately 48 h after transfection, the cells were harvested and used for western blot analysis. Equal amounts of protein (25 µg) were applied on a 7% SDS–polyacrylamide gel. Following electrophoresis, the protein was transferred to a nitrocellulose membrane which was probed with a mixture of rabbit anti-mPGES-1 and anti-COX-2 antibodies using a 1:500 dilution (Cayman, Ann Arbor, MI) and then stained with horseradish peroxidase-labeled goat-anti rabbit antibody using a Chemiluminescence kit (Amersham, England, UK). The numbers on the left represent the molecular mass (in kDa) of the proteins described.