expression vector (Invitrogen) were selected using Zeocin (0.5 mg/ml). To release soluble 7BP anchored on the cell surface, we treated cells with thrombin (10 IU/ml) for 3 days under serum-free conditions. The cleaved 6-His-and FLAG epitope-tagged recombinant 7BP in serum-free conditioned media was purified by sequential nickel and anti-FLAG affinity chromatography. For antibody production, 5 mg of purified 7BP emulsified in Freund's adjuvant were injected into rabbits (Strategic BioSolutions

more »

... c., Ramona, CA). Western blot analysis indicated that the antibody to 7BP does not recognize recombinant LGR8 proteins. . 17. Eight-week-old female mice (strain C57/B6) were treated with equine chorionic gonadotropin (CG) (10 IU, National Hormone and Peptide Program) for 48 hours before ovulation induction with human chorionic gonadotropin (hCG, 10 IU). Mice were mated overnight after ovulation, and pregnant mothers were treated with purified 7BP (500 g, once per day) for 4 days starting from postcoitus day 17. Parturition was monitored at 4-to 6-hour intervals. Five animals per treatment group were used. The pregnancy durations for control and 7BP-treated animals were 508.8 Ϯ 9.0 hours and 536.0 Ϯ 9.1 hours, respectively. The nipple sizes (length ϫ width, mm 2 ) for control and 7BP-treated mice were 1.50 Ϯ 0.05 and 1.07 Ϯ 0.04, respectively.