Total RNA (10 ^g) was loaded on a 1% agarose-2.2 M formaldehyde gel, transferred to nylon membranes (Magna; Micron Separations Inc., Westboro, MA), and then cross-linked by UV irradiation. After 4 h of prehybridization in a 50% formamide solution at 42°C, membranes were sequentially hybridized overnight at 42°C in the same solution containing [a-32P]dCTP-labeled cDNA probes corresponding to human PGHS-2 mRNA and [a-32P]dCTP-labeled 18S ribosomal DNA. Membranes were washed and exposed to x-ray films with an intensifying screen at -80°C for 4-72 h. Membranes were stripped for 30 min in 50% formamide, 6-strength saline-sodium phosphate-EDTA at 65°C between hybridizations. Signals were quantitated by densitometry of the autoradiograms using NIH (Bethesda, MD) Image 1.57 software.
Western Blot Analysis
Cells were rinsed with PBS and then lysed in a homog-enization buffer (50 mM Tris, pH 6.8, 1% SDS, 10% glycerol, 1% (3-mercaptoethanol). For immunoblot analysis, 100 ^g of protein from cell lysates was separated by SDS-PAGE. Proteins were transferred onto polyvinylidene fluoride membranes (Micron Separations Inc.). Membranes were blocked in 5% nonfat milk-Tris-buffered saline with 0.1% Tween 20 (TBST) before incubation with rabbit antibody to either human PGHS-1 (dilution 1:5000), PGHS-2 (dilution 1:2500), or cPLA2 (dilution 1:500) or mouse monoclonal antibody to PGI synthase (dilution 1:5000). Blots were incubated with donkey anti-rabbit IgG (PGHS-1, PGHS-2, and cPLA2) or sheep anti-mouse IgG (PGI synthase) peroxidase-linked secondary antibody in TBST. Chemiluminescent detection was performed using reagents from Dupont, and bands were visualized on Kodak (Eastman Kodak, Rochester, NY) films.
All experimental data are presented as the mean ± SEM. Statview 4.51 (Abacus Concepts, Berkeley, CA) statistical package for the Macintosh was used for all analyses. For Northern blot data, the Student’s t-test was used to determine the level of significance for differences between two treatment (steroid hormones or PGHS inhibitors versus control) groups. Differences in dose response for each treatment were analyzed by ANOVA followed by Fisher’s least-significant-difference procedure. The level of statistical significance was set at p < 0.05.