A volume of sample equivalent to 50 µg of proteins was labeled for DIGE analysis. The biological replicates of each sample were split and marked half with CyDye 3 fluorochrome and half with CyDye 5. The CyDye 2 fluorochrome was added to the internal standard, which is a mixture of all samples in equal amount. The labeling was done by the addition of 400 pmol of dye, followed by a 30 min incubation on ice in the dark. Then, 1 µL of lysine 10 mM was added to stop the reaction and the samples were incubated 10 more min in the same conditions. The samples were combined as follows: 1 Cy3-labeled, 1 Cy5 labeled and 1 internal standard. They were then loaded on strips (pH 3-10 nonlinear, 24 cm) for the first dimension, using the passive rehydration method. Nine µL of ampholytes and 2.7 µL of destreak reagent were added to 450 µL of the sample in buffer solution [urea 7 M, thiourea 2 M, Tris 30 mM and CHAPS 0.5% (w/v)]. The strips were rehydrated with the samples overnight. The isoelectric focusing (IEF) was performed with an Ettan IPGphor 3 system (GE Healthcare). A gradual increase of the voltage was used to reach a total of ca. 90000 V h within 25 h, through 5 steps planned as follows: 0–3 h 100 V, 3–7 h ramping to 1000 V, 7–14 h 1000 V, 14–20 h ramping to 10000 V and 20–25 h 10000 V. The second dimension was run on precast 12% flatbed gels (25×20 cm) in a horizontal electrophoresis tower (HPE FlatTOP Tower, Serva) following the manufacturer’s instructions. The 2D gels were scanned using a laser scanner (Typhoon FLA 9500, GE Healthcare) and consequently analyzed with the software SameSpots (http://totallab.com/home/samespots/).

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