Rat Granulosa Cell Isolation and Culture
Immature female Sprague-Dawley rats (age, 24-25 days) from Charles River Canada (Montreal, PQ) were injected with eCG (15 IU i.p.), and ovaries were collected 24 h thereafter in RPMI 1640 medium supplemented with Hepes (10 mM; pH 7.4) and 10% (w/v) fetal bovine serum (FBS). Granulosa cells were harvested by follicle puncture as previously described, washed, and centrifuged (900 X g, 10 min). Next, 6 X 105 viable cells were plated (6-well plate, Falcon; Becton Dickinson) for 24 h in RPMI 1640 medium with 10% (w/v) FBS under a humidified atmosphere of 95% air and 5% CO2 and cultured for various durations in serum-free medium with adenoviral LacZ or antisense or sense XIAP cDNA. order zyban online
Adenoviral Infection in Primary Granulosa Cell Culture System
Infection of primary granulosa cells with adenoviral XIAP sense and antisense cDNA was performed as described by Kim et al.. Briefly, 1 million granulosa cells were plated in 60-mm dishes for 24 h and infected with adenoviral sense or antisense full-length XIAP or LacZ at an MOI of 5 or 20, respectively. The FSH was added to culture medium 24 h after viral infection, and granulosa cells were cultured for another 24 h. At an MOI of 10, the LacZ infection efficiency over 48 h (as determined by X-gal assay) was more than 90%, and changes in XIAP expression were confirmed by Western blot analysis.
Protein Extraction and Western Blot Analysis
Assessment of XIAP protein contents was performed according to the immunoblotting procedures described by Kim et al. with minor modifications. Briefly, cultured follicles were harvested and lysed mechanically in RIPA buffer (1X PBS [pH 7.4] containing SDS [0.1%, w/v], sodium deoxycholate [0.5%, w/v], NP-40 [1%, v/v], and the protease inhibitors PMSF [1 mM], aprotinin [10 ^g/ml], and sodium orthovanadate [1 mM]). The follicular lysate and harvested granulosa cells were sonicated (three times for 10 sec each on ice) in RIPA lysis buffer. Sonicates were pelleted by centrifugation (14 000 X g, 30 min, 4°C), and the supernatant was retained and stored at -20°C. Protein content of the extracts was determined with the Bio-Rad DC Protein Assay Reagent. Samples were mixed with loading buffer, resolved by 10% SDS-PAGE, and electrotransferred (30 V overnight or 80 V for 2 h) onto nitrocellulose membranes using the Bio-Rad Trans-Blot system.