2-D DIGE was performed as previously described [26 (link), 59 (link)]. Briefly, protein lysates (100 μg) were cleaned using a 2D Clean-up Kit (GE Healthcare) and the precipitated protein was resuspended in UTC buffer. Samples were then labeled with cyanine dyes (CyDye, GE Healthcare). 40 μg of sample was mixed with IEF buffer (8 M urea, 2 M thiourea, 4% w/v Chaps, 10 mM EDTA pH 8.0, 250 mM DTT, plus ampholytes) and samples were isoelectrically focused on nonlinear 18 cm immobilized pH gradient (IPG) 4–7 pH or 7–11 pH strips (GE Healthcare) using the standard protocols. After focusing, samples were equilibrated in 1% DTT (w/v) dissolved in IEF Equilibration buffer (IEF-EQ; 6 M Urea, 5% SDS (w/v), 30% glycerol (v/v)), then in 2.5% iodoacetamide (w/v) dissolved in IEF-EQ. The second dimension electrophoresis was run on a 12% SDS resolving gel. Gels were imaged using a Typhoon 9410 Imager (GE Healthcare) with Cy3 (532-nm laser), Cy5 (633-nm laser) and Cy2 (488-nm laser). Images were analyzed using PDQuest version 8 advanced (Biorad).