Fig. 1.

Analyses of MPGES-1 in three prostate cancer cell lines. (A) For Western blot cells growing on dishes were lysed with M-PER buffer, and 37.5-μg aliquots of total cellular protein were applied to SDS/PAGE gels. For loading controls (β-actin), 7.5-μg aliquots were analyzed. After electroblotting, membranes were incubated with antibodies to MPGES-1 and β-actin (see Materials and Methods). (B) Conversion of PGH₂ to PGE₂ by microsomal proteins prepared from three prostate cancer cell lines. Cells were detached by addition of trypsin/EDTA for 10 min at 37 °C and were collected by centrifugation. After sonication, membrane-bound proteins (microsomes) were prepared (see Materials and Methods). Aliquots (25 μg) in 100 μL were incubated with PGH₂ (10 μM) for 1 min on ice. After solid-phase extraction, samples were analyzed by HPLC-UV (195 nm). Activity is given as picomoles of PGE₂ formed per 25 μg of protein per minute ± SE (n = 3).