Statistically significant (t-test, p,0.05) differentially expressed proteins spots identified in regular 2DE and 2D-DIGE experiments were selected for further MS analysis to establish protein identity. GelCode Blue stained preparative gels containing much higher amount of protein (1 mg) were used for excision of the spots of interest specified in the 2D-DIGE experiment. Spot excision was performed manually. In-gel digestion of the proteins separated by 2D gel electrophoresis was performed as described by Shevchenko et al., with slight modifications [39] . In short, gel slices were cut into small cubes (,161 mm) and washed with 50 mL of stain removal solution (25 mM ammonium bicarbonate buffer) for removal of CBB stain. After washing, 50 mL of 25 mM ammonium bicarbonate/acetonitrile (1:1 v/v) was added, followed by 5 min incubation with occasional vortexing at room temperature. After incubation, the solutions were removed. These two steps are repeated for three times. Then, 50 mL reduction solution (10 mM DTT in 100 mM ammonium bicarbonate) was added and the gel pieces were incubated for 60 mins at 56uC in an air thermostat. Tubes were allowed to cool to room temperature after incubation, and 50 mL of 25 mM ammonium bicarbonate buffer was added to wash the gel pieces followed by dehydration with 25 mM ammonium bicarbonate/acetonitrile (1:1, v/v). After this step, alkylation solution (50 mM IAA in 100 mM ammonium bicarbonate) was added and the tubes containing the gel pieces were incubated for 30 mins at room temperature in dark. Rehydration and dehydration steps were performed twice and gel pieces were allowed to dry. Once the gel slices were properly dried, trypsin solution (Trypsin Gold; Promega, Madison, Wisconsin, United States) was added to the gel pieces keeping the ratio of trypsin: protein around 1:10 (w/w) and incubated at ice for 30 mins for absorption of the solution. After this step, the tubes were incubated overnight at 37uC. Adequate amount of ammonium bicarbonate buffer was added to cover the gel pieces. Extraction of the digested peptides from the gel matrix was performed using 100 mL of extraction buffer (0.2% formic acid in 66% acetonitrile) after completion of the enzymatic reaction. Extraction step was repeated thrice to ensure maximum recovery of the digested peptides. The collected supernatants were pooled in a single tube and concentrated using speed vac. After extraction trypsin digested samples were further processed using Zip-Tip C18 pipette tips (Millipore, USA) according to the manufacturer's protocol for removal of salts and enrichment of the peptides.