Two-dimensional gel electrophoresis was performed as previously reported with minor modifications (9, 10). Briefly, protein from cells (1 × 106) was isolated using TRIzol reagent (Invitrogen) and dissolved in 150 μl of rehydration buffer [8 M urea, 2% CHAPS, 50 mM dithiothreitol (DTT), 0.5% immobilized pH gradient (IPG) buffer, and 0.002% bromophenol blue]. After centrifugation at 20,000g for 30 min, the supernatant was loaded on Immobiline DryStrips (GE Healthcare) for isoelectric focusing (IEF) with a program comprising 20 V, 12 hours (rehydration); 300 V, 1 hour; 1000 V, 1 hour; 2000 V, 1 hour; 5000 V, 3 hours; and 5000 V, 4 hours. After IEF, strips were incubated with SDS equilibration buffer [50 mM tris-HCl (pH 8.8), 8 M urea, 30% glycerol, 2% SDS, and 0.001% bromophenol blue] containing 70 mM DTT for 15 min and then SDS equilibration buffer containing 2-iodoacetamide (25 μg/ml) for 15 min. Strips were washed with SDS-PAGE buffer, resolved by SDS-PAGE, and analyzed by immunoblotting.

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