2 de manual

Approximately 1,300 µg of the protein samples were diluted to a volume of 450 µL with lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 13 mM DTT), followed by loading onto a 24-cm IPG strip (immobilized pH gradient) with a linear pH gradient of 4–7 (GE Healthcare, Uppsala, Sweden). The strips were hydrated for 18 h at room temperature, and then subjected to IEF on an Ettan IPGphor isoelectric focusing system, following the instructions of the manufacturer (2-DE Manual, GE Healthcare), with some modifications as described previously^{22 (link)}. After IEF, the IPG strips were equilibrated first in an equilibration solution containing 1% DTT, followed by an equilibration solution with 4% iodoacetamide. The strips were then transferred to an Ettan Dalt system (GE Healthcare) to perform SDS-PAGE using 12.5% SDS polyacrylamide gels^{47 (link)}.
The gels were visualized via the GAP staining method^{48 (link)} and scanned with ImageMaster Labscan V3.0 (GE Healthcare, Uppsala, Sweden), and image analysis was performed with the ImageMaster 2D Platinum software package (GE Healthcare, Uppsala, Sweden). Spots that were present in all replicate gels, showing Student’s t test p-values <0.05 and a relative fold change of at least a 1.5 in their quantity, were further analyzed.

2-DE was performed on an Ettan IPGphor isoelectric focusing system according to the manufacturer’s instructions (2-DE Manual, GE Healthcare, Uppsala, Sweden). The 24 cm IPG strips (immobilized pH gradient) with a linear pH gradient of 4–7 (GE Healthcare) were used, approximately 1,300 µg protein samples were loaded on, and 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gels were used for SDS- polyacrylamide gel electrophoresis (SDS-PAGE). Each protein extracts were performed on 2-DE gels in triplicate for technical replicates. The experimental procedures were as previously described^{30 (link)}.
Gels were stained using a GAP staining method^{46 (link)} and scanned with the ImageMaster Labscan V3.0 (GE Healthcare). Image analysis was conducted using a ImageMaster 2D Platinum software package (GE Healthcare). Only the spots that were present in all replicate gels and shown a Student’s t test p-value < 0.05 and a relative change in quantity of at least 1.5-fold in their quantity, were considered as DAPs for further analysis^{30 (link)}.