Figure 5. Effects of DN-CK-II on cPGES phosphorylation and function.

(A) Expression levels of PGE₂-biosynthetic enzymes in mock- or DN-CK-II-transfected 3Y1 cells, as assessed by immunoblotting. (B) Association of cPGES with Hsp90 (top panel) and phosphorylation of cPGES (middle panel) in mock- or DN-CK-II-transfected cells. cPGES–Hsp90 association was assessed by immunoprecipitation (IP) and subsequent SDS/PAGE and immunoblotting, and cPGES phosphorylation was assessed by immunoprecipitation from replicate ³²P-labelled cells, followed by SDS/PAGE and autoradiography. Equal sample loading into each lane was verified by immunoblotting with anti-cPGES antibody (bottom panel). Blots representative of three independent experiments are shown. (C) cPGES enzymic activity in lysates of mock- or DN-CK-II-transfected cells. Values show relative enzymic activity, with the activity of control cells regarded as 1. (D) PGE₂ production by mock- or DN-CK-II-transfected cells treated for 30 min with 10 μM AA. (E) PGE₂ production by A23187-stimulated mock- or DN-CK-II-transfected cells. Cells were treated with (+) or without (−) 10 μM A23187 for 30 min. (F) AA release from mock- or DN-CK-II-transfected 3Y1 cells. Cells were pre-incubated with [³H]AA, and [³H]AA release with (+) or without (−) stimulation with A23187 for 30 min was measured. In (C–F), results are means±S.D. of three to five independent experiments (*P<0.05 compared with replicate mock-transfected cells).