Ief cell

2-DE was performed as described previously with minor modifications (Tong et al., 2008) . Briefly, Cells were treated with 4μM pemetrexed and collected into a 1.5mL centrifuge tube after incubated for 48h. Then cells were re-suspended with lysis buffer (7M urea, 2M thiourea, 4%CHAPS, 50mM DTT, 0.2% Bio-lyte (w/v), 50ug/ml RNase and 200ug/ml Dnase). After mixed and placed at 4℃ for 15min, the samples were centrifuged for 60min at 4℃, 15, 000rpm, the supernatants were harvested and the concentration of protein was measured by a Bio-Rad protein assay kit. The proteins were collected and stored at -80℃. Subsequently IPG strips were taken from frige and maintained at 4℃ for 10min. The protein samples (2mg) were loaded onto IPG strips 17cm, pH3-10, non-liner, Bio-Rad and held for 45min. After IPG strips rehydrated for 12-16h, IPG strips were transformed into IEF cell (Bio-Rad). Composition of rehydration buffer was 7M urea, 2M thiourea, 4% CHAPS, 50mM DTT, 0.2% Bio-lyte (w/v). Once IEF was done, the strips were equilibrated with equilibration buffer containing 50mM Tris-Hcl pH8.8, 6M Urea, 30% glycerol, 2% SDS, 10mM DTT for twice. And then strips transferred to SDS-polyacrylamide gel electrophoresis system with 12% gels. The spots of protein were visualized by Coomassie Brilliant Blue (CBB) R-250. Three independent experiments were repeated.

For the first-dimension electrophoresis, approximately 200 μl of sample from AD and ND subjects were applied to 110-mm pH 3-10 IPG® ReadyStrip (Bio-Rad, Hercules CA). The strips were then actively rehydrated in the protean isoelectric focusing (IEF) cell (Bio-Rad) at 50 V for 18 h. The isoelectric focusing was performed in increasing voltages as follows; 300 V for 1 h, then linear gradient to 8000 V for 5 h and finally 20 000 V/h. Strips were then stored at -80 °C until the 2D electrophoresis was performed. For the second dimension, the IPG® Strips, were thawed and equilibrated for 10 min in 50 mM Tris-HCl (pH 6.8) containing 6 M urea, 1% (w/v) sodium dodecyl sulfate (SDS), 30% (v/v) glycerol, and 0.5% dithiothreitol, and then re-equilibrated for 15 min in the same buffer containing 4.5% iodacetamide instead of dithiothreitol. Linear gradient precast criterion Bis-Tris gels (12%) (Bio-Rad) were used to perform second dimension electrophoresis. Precision Protein™ Standards (Bio-Rad, CA) were run along with the samples at 200 V for 50 min. For the detection of 3-NT modified proteins, the gels were incubated in fixing solution (10% acetic acid, 40% methanol) for 40 min and stained overnight at room temperature with 50 mL SYPRO Ruby gel stain (Bio-Rad). The SYPRO Ruby gel stain was then removed and the gels stored in deionized water.

One milliliter of philibertain g II was treated with 3.5 vol acetone and the precipitate was redissolved with 100 μL of deionized water. Five percent polyacrylamide gels were used, containing alternatively Pharmalyte broad range pH 3-10 (GE Healthcare Life Sciences). A Mini isoelectric focusing (IEF) Cell (Model 111, Bio-Rad) was employed to carry out isoelectric focusing.
Unstained IEF gels were contacted for 10 min in an oven at 60 °C with an agarose gel imbibed with a 1 % casein solution. After incubation, the agarose gel was dehydrated and stained by Coomassie Brilliant Blue R-250. Unstained bands evidence proteolytic activity [30] .