Fig. 2.
The enzyme activity and respective protein expression of eicosanoid biosynthesis in cultured OEC. a & b: HPLC analysis of 14C-labelled eicosanoids generated in Control and AdPGIS-transduced OEC cultures, respectively, in response to [1-14C] arachidonic acid (AA). 6-KP denotes 6-keto-PGF1α, the product of PGI2 hydrolysis. Each prostanoid peak was verified by coelution with an authentic radiolabelled prostanoid, c: Western blot analysis of protein levels of PGIS, COX-1 and COX-2 in cultured OEC receiving AdPGK or AdPGIS infection. Measurement of eicosanoid biosynthesis and biosynthetic enzyme expression in cultures was conducted at 3 days after Ads transduction. Confluent OEC cultures were pulsed with [1-14C] AA for 10 min. The released [1-14C] eicosanoids in the released medium of OEC were purified and subsequently analyzed by HPLC with a radiochemical flow-through detector. Note that OEC constitutively expressed COX-1 and COX-2 but not PGIS. On Ad-PGIS infection, PGIS expression was increased and AA metabolites in OEC were shunted through 6-ketoPGF1α (6-KP) synthesis