Criterion xt gel

Rat liver tissue was homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors (Roche). The protein concentration was determined using Micro BCA (Pierce) according to the manufacturer's protocol. For all of the blots except acetyl-CoA carboxylase (ACC), 30 μg of protein was separated using a 4–12% Criterion XT gel (Bio-Rad). For ACC, 10 μg of protein was separated using a 3–8% tris-acetate Criterion XT gel (Bio-Rad). Protein was transferred using a Bio-Rad Western blotting transfer system at 100 V for 1 hr onto nitrocellulose membranes. The membranes were blocked using SuperBlock T20 Tris-buffered saline (TBS) blocking buffer. Primary antibody incubations were diluted as indicated in Supplemental Table 4 in Tris-buffered saline with 0.1% Tween 20 (TBST) with 3% non-fat dry milk except for the ACC antibody, which was incubated in 5% BSA in TBST overnight. The membranes were washed in TBST and incubated with the appropriate secondary antibody at the indicated dilutions (Supplemental Table 4) in TBST with 3% non-fat dry milk or 5% BSA for 1 hr. The membranes were incubated with enhanced chemiluminescence Western blotting substrate and bands were visualized using a Fujifilm LAS 4000 imager (Fujifilm) and Multi Gauge V3.2 software (Fujifilm).