The separated proteins were electrophoretically transblotted to a 0.2-��m nitrocellulose membrane (no. LC2000; Invitrogen) at 250 mA for 3 h in transfer buffer Bioactive compound (25 mM Tris-HCl, 192 mM glycine, 20% methanol, pH 8.3). The membrane was washed in TBS (25 mM Tris-HCl, 137 mM NaCl, pH 7.5) and incubated in blocking buffer (5% nonfat dry milk in TBS-T) for 1 h at room temperature. After blocking buffer incubation, the membrane was washed three times in TBS-T (0.1% Tween 20 in TBS) for 10 min at room temperature, and then the membrane was cut into two or three pieces. The membranes were then incubated separately with a primary antibody in blocking buffer specific to each protein (Table 2) overnight at 4 C. After primary antibody incubation, the membranes were washed one time for 10 min in blocking buffer at room temperature, and washed two times for 10 min in TBS-T.

After washing, the membranes were incubated with secondary antibody in TBS-T (anti-rabbit Ig, HRP-linked whole antibody produced in donkey [Amersham Biosciences, San Francisco, CA, USA; no. NA934; 1: 4000] for COX-1, COX-2, PGFS and PGES protein; anti-goat, HRP-linked whole antibody produced in donkey [Santa Cruz Biotechnology, CA, USA; no, sc-2020; 1:4000] for CBR1 protein; anti-mouse, HRP-linked whole antibody produced in sheep [Amersham Biosciences Corp.; no. NA931; 1: 40000] for beta-actin protein) for 1 h at room temperature and washed three times in TBS for 10 min at room temperature. The signal was detected using an ECL Western Blotting Detection System (no. RPN2109; Amersham Biosciences). Table 2.

Primary antibodies for Western blotting The intensity of the immunological reaction in the cells was estimated by measuring the optical density in the defined area by computerized densitometry using NIH Image (National Institutes of Health, Bethesda, MD, USA). PG and P4 determination The conditioned medium was collected in 1.5 ml tubes containing 5 ��l of a stabilizer solution (0.3 M EDTA, 1% (w/v) acid acetyl salicylic, pH 7.3). The concentrations of PGF and PGE2 in the culture medium were determined by EIA [25]. The PGF standard curve ranged from 0.016 to 4 ng/ml, and the ED50 of the assay was 0.25 ng/ml. The intra- and interassay coefficients of variation were on average 2.8 and 7.7%, respectively. The PGE2 standard curve ranged from 0.039 to 10 ng/ ml, and the ED50 of the assay was 0.

625 ng/ml. The intra- and interassay coefficients of variation were on average GSK-3 11.3 and 13.3%, respectively. The concentrations of P4 in the culture medium were determined by EIA [25]. The P4 standard curve ranged from 0.391 to 100 ng/ml, and the ED50 of the assay was 0.09 ng/ml. The intra- and interassay coefficients of variation were on average 5.3 and 7.9%, respectively. Cell viability test The cell viability was determined using a Dojindo Cell Counting Kit including WST-1 (no. 345-06463; Dojindo, Kumamoto, Japan) as described previously [11].