**2.6 2-DE analysis**

2-DE was performed using IPGphor and Multiphor (GE healthcare). Briefly, the proteins were resuspended in 150 μL of a 2-DE sample buffer containing 9 M urea, 65 mM DTT, 2% Pharmalyte (GE Healthcare), 4% CHAPS, and 1% bromophenol blue and then centrifuged at 4ºC and 23000g for 30 min to remove debris. The supernatant was then mixed with a rehydration buffer consisting of 8 M urea, 4% CHAPS, 0.5% IPG buffer 3-10 NL (GE Healthcare), 19 mM DTT, and 5.5 mM Orange G to a final volume of 350 μL. The first dimension was performed by in-gel rehydration for 12 h in 30 V on 18 cm pH 4-7 linear or pH 3-10 nonlinear IPG strips (Immobiline DryStrips, GE Healthcare). The proteins were then focused at 53000 Vh at a maximum voltage of 8000 V (Görg et al., 2000). The second dimension (SDS-PAGE) was performed by transferring the pI focused proteins (IPG strips) to homogeneous or gradient home-cast gels on gel bonds. The electrophoresis was performed at 40-800 V, 10°C, 20-40 mA, overnight.
