**2.5 IEF and 2-DE**

Two-hundred uL of each sample was placed onto individual wells in IPG rehydration trays from Proteome Systems (Woburn, MA, USA). Bio-Rad ReadyStrip IPG strips with a pH range of 3-10, 4-7, or 7-10 (Hercules, CA, USA) were placed onto each sample, and the tray was placed into a humidifying Ziploc bag. Rehydration occurred over six hours until all the sample was visibly absorbed by the strip. At the termination of rehydration, strips were placed into isoelectric focusing trays and ran at 10,000 volts (maximum voltage) for 110,000 accumulative volt-hours. Strips were equilibrated twice for 10 minutes in 375 mM Tris-HCl containing 2.5% SDS, 3M urea, 0.01% and phenol red, then placed onto Criterion Tris-HCl 8- 16% IPG+1 gels (Bio-Rad Laboratories, Hercules, CA) and ran at 120 V and 60 mA/gel. 2-DE gels were ran for all IPGs, only 4-7 gels are shown.

#### **2.6 Digital image analyses**

The 24-bit images were analyzed using PDQuest™ software (Bio-Rad, v.7.1). Background was subtracted, and protein spot density peaks were detected and counted. A reference pattern was constructed from one of the individual gels to which each of the gels in the matchset was matched. Numerous proteins that were uniformly expressed in all patterns were used as landmarks to facilitate rapid gel matching. After matching, the total spot count was determined for each gel.
