Autoradiograph film

Detection of UCP2 was performed by western blotting. Briefly, organs were perfused with 20 mM ethylenediaminetetraacetic acid (EDTA, pH 7.4). Liver tissues cut into small pieces were homogenized at 4°C in lysis buffer containing protease inhibitors (Roche, AG, Basel, Switzerland). Periepididymal adipose tissues were homogenized at 4°C in RIPA buffer with protease inhibitors (Roche, AG, Basel, Switzerland) and phosphatase inhibitor cocktails (Roche). Tissues were stored at -20°C for further protein quantification. Western blot analyses were performed with whole liver and adipose tissues lysates (40 μg of proteins) using anti-UCP2 (1:1000 dilution, Abcam) and anti-β-actin (1:15000 dilution, Sigma). Detection was performed with the Super Signal Chemiluminescence kit (Pierce), exposing the membrane to an autoradiograph film (GE Healthcare). Bands were digitalized and analyzed by size and intensity by the Image Master 2D Elite program. Anti-AMPK (Cell Signaling) was used, and infrared-labeled goat anti-mouse IRDye 800CW secondary antibodies (Li-Cor Biosciences) were added to bind to the primary antibody. Detection was performed with an Odyssey scanner. Bands were digitalized and analyzed by size and intensity by the Image Studio 3.1 [30 (link)].

Detection of PPAR gamma was performed as previously described [22 (link)] with minor modifications. Briefly, we perfused organs with 20 mM ethylenediaminetetraacetic acid (EDTA) pH 7.4. We cut liver tissues into small pieces and mixed with lysis buffer (with a cocktail of protease inhibitors) at 4°C in (Complete, Roche AG, Basel, Switzerland). We lysed periepididymal adipose tissues at 4°C in RIPA buffer with protease inhibitors (Roche AG, Basel, Switzerland) and phosphatase inhibitor cocktail (Roche). We stored tissues at −20°C for further protein quantification by BCA. Western blot analysis was done with whole liver and adipose tissue lysates (40 μg of proteins) using anti-PPAR gamma (1 : 1000, Santa Cruz) and anti-β-actin (1 : 15000 dilution, Sigma), and detection was performed with the “SuperSignal Chemiluminescence” kit (Pierce), after exposing the membrane to an autoradiograph film (GE Healthcare). Bands were digitalized and quantified by the ImageMaster 2D Elite program.