Activin-lnduced Inhibin: MATERIALS AND METHODS(2)

10 Dec

GC Culture

Rats were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the University of Califomia-San Diego (UCSD) Animal Subjects Committee. GC were collected from ovaries of immature DES-primed rats and plated into 12-well dishes (Coming Glass, Coming, NY) that had been serum-coated. Cells (1 X 106 viable cells/ ml) were cultured in serum-free tissue culture medium (McCoy’s 5a medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 |xg/ml streptomycin sulfate, and 100 nM androstenedione). Cells were cultured at 37°C in a humidified atmosphere of 95% air:5% C02 with or without the indicated concentrations of test samples. birth control pills

Western Immunoblot Analysis

Cell-free supematants (conditioned medium) were centrifuged (500 X g for 5 min) and concentrated 15 times using the Ultrafree-MC Filter Unit (10 000 Mr cut-off; Mil-lipore, Bedford, MA). Samples were diluted with reducing buffer (5% (3-mercaptoethanol), heated at 85°C for 3 min, and loaded onto 14% Tris-glycine polyacrylamide gel using the Xcell mini-cell system (Novex, San Diego, CA). Following electrophoresis, performed at a constant voltage of 125 V for 1.5 h, the protein samples were transferred onto a 0.45-|xm nitrocellulose membrane (Bio-Rad, Hercules, С A) for 2 h at 150 mA using the Mini Blot module (Novex). Molecular weights were estimated by comparison with known molecular weight standards (Bio-Rad). Im-munostaining was performed by incubating overnight at 4°C with Inh-a antibody (1:2000 dilution in 1% casein/ TBST [50 mM Tris-HCl, 150 mM NaCl with 0.1% Tween 20]). Membranes were washed three times with TBST and then incubated with peroxidase-conjugated goat anti-rabbit IgG (diluted 1:10 000 in casein/TBST buffer) for 2 h. The membranes were washed and incubated with enhanced chemiluminescence (ECL) detection reagents (DuPont, Wilmington, DE) for 1 min and exposed to a Hyperfilm ECL (Amersham) for 1-3 min.