Figure 3.

(A) cPLA_2α activity assay: for the in vitro cPLA_2α activity assay, small unilamellar vesicles containing 1-palmitoyl-2 arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-palmitoyl-2-oleoyl-sn-glycerol (POG) 2:1 were prepared and incubated with cPLA_2α enzyme isolated from Sf9-insekt cells. The IC₅₀ value was calculated using a sigmoid Emax model. Data presented are means ± S.E.M. of four independent experiments; (B) PGE₂ in cell supernatant of HeLa cells with or without arachidonic acid addition: HeLa cells were stimulated with IL-1β (1 ng/mL) and TNFα (5 ng/mL) and simultaneously treated with R-flurbiprofen (10, 20, 50 µM), 10 µM celecoxib, 10 µM SC-560 or 5 µM cPLA_2α inhibitor. Additionally, 20 µM arachidonic acid (AA) was added to the cells, and after 16 h incubation, the PGE₂ level in supernatants was determined. Means ± S.E.M. of three independent experiments; (C) Western blot analysis of cPLA_2α in A549 cytosolic and membrane fractions: A549 cells were stimulated with the Ca²⁺-ionophore (A23187) for 10, 30 or 60 min and additionally treated with 100 µM R-flurbiprofen. Actin was used as a loading control. One representative experiment out of three is shown, as well as the quantification of the three Western blots. Data presented are means ± S.E.M. of three independent experiments; statistical analysis was done with one-way ANOVA. Significant p values are shown as: ** p ≤ 0.01, * p ≤ 0.05.