Two-DE analysis was carried out as previously described [21, 22]. In brief, one-dimensional isoelectric focusing (IEF) gel electrophoresis was performed using IPG gel strips (pH 4–7; 7 cm; GE Healthcare, WI). Approximately 1000 μg of protein from each group was incubated with the IPG strips and run at 50 V for 6 h, at 100 V for 6 h, and finally at 2000 V for 6 h. After IEF gel electrophoresis, the IPG strips were equilibrated for 15 min in equilibration buffer [50 mM Tris-HCl, pH 8.8, 6 M urea, 1% SDS, 30% (v/v) glycerol, and 1% (w/v) DTT] and then for 15 min in equilibration buffer containing 2.5% (w/v) iodoacetamide instead of DTT. For the second dimension, the equilibrated IPG strips were transferred onto 15% SDS-PAGE gels at 5 mA/gel for 7 h.