Quantitative results obtained by densitometric analysis of the low-molecular-weight DNA fragments represent the mean 6 SEM of three independent gel runs.
One hundred follicles per ovary were resuspended in 5 volumes of lysis buffer (20 mM Tris-HCl [pH 8], 137 mM NaCl, 1% NP-40, and 10% glycerol) supplemented with protease inhibitors (0.5 mM PMSF, 0.025 mM N-CBZ-l-phenylalanine chloromethyl ketone, 0.025 mM N-p-tosyl-lysine chloromethyl ketone, and 0.025 mML-1-tosylamide-2-phenyl-ethylchloromethyl ketone) and homogenized with an Ultra-Turrax (IKA Werk, Breisgau, Germany) homog-enizer.
Samples were centrifuged at 4°C for 10 min at 10000 X g, and the resulting pellets were discarded. Protein concentration in the supernatant was measured by the Bradford assay. After boiling for 5 min, 40 ig of protein was applied to a 12% SDS-polyacrylamide gel, and electrophoresis was performed at 25 mA for 1.5 h. The resolved proteins were transferred for 2 h onto nitrocellulose membranes. The blot was preincubated in blocking buffer (5% nonfat milk and 0.05% Tween-20 in 20 mM triethanolamine-buffered saline [TBS; pH 8.0]) for 1 h at room temperature and incubated with appropriate primary antibodies (TNFRSF6 1/500, FASLG 1/500, BAX 1/200, BCL2 1/500, and BCL2L1 1/200 in TBS) in blocking buffer overnight at 4°C.