Effect of TNFα on protein expression of PGE synthases and COX-2 and production of PGE
. (A) Gingival fibroblasts were treated with TNFα (20 ng/ml) or without TNFα for the indicated time period. Cells were harvested, total protein was extracted and protein expression of the PGE2-synthesizing enzymes mPGES-1, mPGES-2, cPGES and COX-2 as well as the loading control actin was analyzed by western blot. (B) Gingival fibroblasts were stimulated with TNFα (20 ng/ml) or cultured without TNFα (control) for 24 h. Protein expression of mPGES-1, mPGES-2, cPGES and COX-2 was investigated by flow cytometry, using specific antibodies. (C) Gingival fibroblasts were cultured with or without TNFα (20 ng/ml) for the indicated time period. Levels of PGE2 in the culture media were measured by EIA on a Luminex system, and are presented as mean ± s.d. Asterisks (*) indicate a significant difference from untreated cells at the corresponding time point (p < 0.05). The results are representative for all three cell lines and all analyses were performed in triplicates.