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Equal amounts of protein were diluted in sample reducing buffer containing 5% ��-mercaptoethanol and heated at 95��C for 5 minutes. Proteins were separated using 12% polyacrylamide gels at 60 V for 30 minutes. Blotting of protein was performed on polyvinylidene difluoride membranes using a Trans-Blot (BioRad) apparatus at 350 mA for 1 hour at 4��C. Membranes were blocked with 5% fat-free milk in 0.1% Tween-20 phosphate buffered saline (PBS-Tween) at room temperature. Primary antibodies for AMPK��1 (PA1-2109, 1:5,000; Thermo Fisher Scientific) and p-AMPK��1 (PA5-17831, 1:1,000; Thermo Fisher Scientific) were incubated overnight at 4��C. Blots were washed with PBS several times and then incubated with secondary antibody (goat anti-rabbit IgG-HRP, 1:6,000 and 1:2,000; learn more Thermo Fisher Scientific) for 1 hour at room temperature. Blots were washed with PBS several times and then SuperSignal West Femto substrate (34095; Thermo Fisher Scientific) was added lazabemide for 5 minutes. Chemiluminescence was detected using an Alpha Innotech Imager (ProteinSimple, Santa Clara, CA, USA). Density of the bands was quantified using the Alpha Innotech software. Statistical analyses Data were analyzed in a completely randomized design using general linear models (GLM) procedures of SAS (SAS Institute Inc., Cary, NC, USA) with palmitoleic acid dose level, tissue, and two-way interaction in the model. For serum data, palmitoleic acid dose level, time for experiment, and the two-way interaction were included in the model. Orthogonal contrasts were used to assess linear or quadratic changes in variables because of dose level of palmitoleic acid infusion. For gene expression, delta CT values were analyzed using GLIMMIX procedure with treatment, tissue, and check details two-way interaction in the model. Gene expression levels were calculated using the ����CT methods13 and are presented as fold-change in gene expression for experiment (10 mg/kg lipid weight (LW)/d C16:1) versus controls (0 mg C16:1/kg LW/d). Results Infusion of 10 mg/kg BW/d palmitoleic (C16:1 cis-9) acid intravenously in obese sheep reduced (P0.05) by palmitoleic acid infusion for 28 days. The lambs from this experiment averaged 1.95 cm of fat thickness with a body composition of 40% of total lipid, which would be considered obese. Sheep are more similar to humans in fat distribution patterns than other animal models. Sheep deposit more fat within the abdominal cavity (about 33% of total fat) than pigs or rodents.13 Table 2 Gain, carcass traits, and body composition of lambs infused with varying levels of palmitoleic (C16:1) acid Serum palmitoleic (C16:1 cis-9) levels increased (P
