Alpha imager 5500

Tissue lysates from the intraperitoneal adipose tissue or the aortae (30 µg) were resolved by SDS-PAGE, and proteins were then transferred to polyvinylidene difluoride membranes. After blocking with 5% milk blocking buffer (Tris-buffered saline with 0.1% Tween 20), the membranes were incubated overnight at 4°C with antibodies against lipocalin-2 and β-actin (Cell Signaling, Beverly, MA, USA), respectively, and followed by horseradish peroxidase-conjugated secondary antibody. Detection of immunoreactive band was achieved using enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China), and scanned on an Alpha Imager 5500 (Alpha Innotech, San Leandro, CA, USA) imaging densitometer. The expression of proteins was normalized to that of β-actin.

To obtain tissue lysates, eWAT and sWAT were homogenized in 10% sodium dodecyl sulfate buffer containing protease and phosphatase inhibitors. An antibody targeting PR domain containing 16 (PRDM16) (Abcam) was used for western blotting. Bands were detected with ECL enhanced chemiluminescence detection reagents (Applygen, Beijing, China) and scanned with an Alpha Imager 5500 (Alpha Innotech, San Leandro, CA, USA) imaging densitometer.

Cells were lysed after the indicated treatments. Approximately, 5 mg of total protein for each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk and incubated with appropriate primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The immunostaining signals were visualized using the ECL plus reagent (Applygen, Beijing, China), imaged, and analyzed with an Alpha Imager 5500 (Alpha Innotech, San Leandro, CA).