Testes from normal 10-week-old mice (n = 3) were lysed by sonication in isoelectric focusing rehydration buffer (7 M urea; 2 M thiourea; 4% CHAPS; 100 mM DTT; 0.2% Bio-Lyte, pH 5–8; 0.01% bromophenol blue; and protease inhibitor). Insoluble material was pelleted (12,000 g, 15 min and 100,000 g, 60 min) and the resulting supernatant was desalted using a 2D Cleanup Kit (GE Healthcare UK Ltd, Buckinghamshire, England). One hundred microgram of protein in a total of 200 μl of rehydration buffer was applied to 24-cm pI gradient strips (pI4–7 and pI6–9; GE Healthcare UK, Buckinghamshire, England) for overnight rehydration. First-dimension isoelectric focusing was preformed on a GE Multiphor II System (GE Healthcare UK). After focusing, strips were equilibrated with equilibration buffer (6 M urea; 50 mM Tris-HCl, pH 8.0; 2% SDS; 20% glycerol; and 2% w/v DTT) for 15 min. The strips were further equilibrated with equilibration buffer II (6 M urea; 50 mM Tris-HCl, pH 8.0; 2% SDS; 20% glycerol; and 2.5% w/v iodoacetamide) for 15 min and then directly applied to a 7.5% isocratic SDS-polyacrylamide gel for the second dimension separation. The resulting gel was then transferred to a membrane for Western blot or stained using a silver stain kit (Wako Pure Chemical Industries, Osaka, Japan).