Total akt1 2

Western analysis was performed using cell lysates harvested in Mammalian Cell Lysis Buffer (Sigma Aldrich) with the addition of protease and phosphatase inhibitors (Biorad). Proteins (30 µg) were separated using a 10% Criterion-Tris·HCl gel (Bio-Rad, Hercules, CA) and subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Blots were probed with primary antibodies against β-actin (1:1,000; Cell Signaling, Danvers, MA). Total AKT1/2 (1:1,000; Cell Signaling, Danvers, MA), P-AKT1/2 (Ser473) (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), Total IRS-1(1:1,000; Cell Signaling, Danvers, MA), and P-IRS-1 (Tyr612) (1:1,000; Cell Signaling, Danvers, MA) followed by anti-rabbit secondary antibodies (1:10,000; Jackson Immuno-Research Laboratories, West Grove, PA). Proteins were visualized using Super-Signal Chemiluminescent Substrate (Pierce, Rockville, IL) and a ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA). Phosphorylated protein expression is expressed as a value relative to the total form of the protein. All values are corrected to total protein loading using Image Lab software (Bio-Rad, Hercules, CA).