Enhanced chemiluminescence plus reagent

For protein isolation, each 50-mg tissue sample was ground into powder with liquid nitrogen and lysed with 600 µl radioimmunoprecipitation assay (50 mM Tris-base, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100 and 1% sodium deoxycholate) lysis buffer. Following centrifugation at the speed of 12,000 × g at 4°C for 5 min, the supernatant was obtained and the protein concentration was assessed using Pierce BCA Protein Assay kit. (Thermo Fisher Scientific, Inc.) A similar method was applied to extract proteins in ESCs. Isolated proteins (10 µl) were separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The proteins were incubated with primary antibodies for 1 h at room temperature. The primary antibodies were rabbit anti-human PAI-1 (no. 11907; 1:800) and rabbit anti-human GAPDH antibody (no. 2118; 1:2,000). The membranes were then incubated with secondary antibody overnight at 4°C. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (no. 7074; 1:1,000). All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Finally, the membrane was developed by enhanced chemiluminescence plus reagent (EMD Millipore, Billerica, MA, USA). Image Lab™ software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was applied to analyze the blot images and the intensity of the bands.

Total protein was extracted with SDT buffer (4% [w/v] sodium dodecyl sulfate, 0.1M Tris/HCl pH 7.6, 0.1 M dithiothreitol) and quantified with a bicinchoninic acid protein assay kit. Equal amounts of protein (20 μg) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, which was cut into several strips that were blocked and incubated with primary antibodies (Supplementary Table 2). After probing with secondary antibodies, the blots were visualized with Enhanced Chemiluminescence Plus reagent (EMD Millipore Corporation, Billerica, MA, USA).

Tissues and cells were lysed using RIPA lysis buffer [150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholic sodium salt, 50 mM Tris HCl (pH 7.4), 2 mM EDTA, 2 mM Na₃VO₄, 10 mM NaF and one tablet of Roche Complete inhibitor cocktail (Roche Diagnostics) per 30 ml]. The protein concentration was measured using a BCA protein assay kit. Equal amounts of protein (50 µg) from each sample were separated on a 10% Nupage gel, transferred to the nitrocellulose membranes and immunoblotted with primary antibodies overnight at 4˚C. All the primary antibodies were used at a 1:1,000 dilution (antibody: Dilution buffer). The membranes were washed with Tris-buffered saline containing Tween-20 (0.15%, TBS-T), incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000) for 2 h at room temperature, and washed 3 times with TBS-T. The blots were visualized using Enhanced Chemiluminescence Plus reagent (EMD Millipore) and imaged using a GE gel imaging AI600 (Amersham Imager 600; GE Healthcare).

The total proteins were extracted using lysis buffer. Equal amounts of the protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h at room temperature using 5% nonfat milk and were incubated overnight at 4℃ in the appropriate primary antibody, which included rabbit anti-p-ACC (abcam, 1:250), rabbit anti-ACC (abcam, 1:250), rabbit anti-p-AMPK (abcam, 1:250), rabbit anti-AMPK (abcam, 1:250) and rabbit anti-GAPDH (abcam, 1:250). Then, the membranes were washed 3 times for 10 min each with Tris-buffered saline containing Tween and were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Following additional washes, the immunoreactive proteins on the membrane were visualized with enhanced chemiluminescence plus reagents (Millipore, Plano, TX, USA). The intensities of the immunoreactive proteins were measured via image J and were normalized to GAPDH. All the experiments were repeated at least 3 times.