Whole epididymides from each treatment group were powdered in a porcelain mortar with a pestle under liquid nitrogen. The powder was scraped into an Eppendorf tube and stored on dry ice. Protein was extracted by the addition of 200 pl of cold extraction buffer (10 mM Hepes pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF and 1X Complete protease inhibitor cocktail [Roche, Lewes, U.K.]). The tissue remained on ice for 15 min before adding 25 pl of 10% Nonident P-40 (Sigma). The tube was then vortexed three times for 10 sec. The tube was then centrifuged at 12 000 X g for 1 min at 4°C, the supernatant was decanted, and 100-pg aliquots were frozen on dry ice before being stored at —40°C.
Western Blot Analysis
The expression of H+-ATPase and p-actin protein were determined in 25-day-old rat epididymal protein extracts using Western blotting. Protein markers (Bio-Rad Laboratories, U.K.) and protein samples (from all treatment groups) were separated by SDS-PAGE using gradient gels (4%-20%; Novex precast gels, Invitrogen,). The gels were run at 110 V for ~2 h before blotting onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Watford, U.K.) at 33 V for 180 min using Tris/Gly transfer buffer (Invitrogen). The membranes were blocked for 1 h in 5% milk/ TBS, 50 mM Tris-HCl, and 150 mM NaCl. The membranes were washed thoroughly in TBST (TBS containing 0.05% Tween-20; Sigma) before the primary antibody was added. An affinity-purified chicken antiserum against the 31-kDa subunit of the H+-ATPase was added at a dilution of 1:1000 (TBS containing 0.02% sodium azide) and incubated at 4°C overnight.