Western Blot Analysis of Thermogenic Proteins

Proteins were extracted from cell lysates following the manufacturer’s protocols (Beyotime, China). Protein concentration was quantified using the BCA protein assay kit (Thermo Fisher Scientific, United States) and 30 μg protein was separated in a 12% SDS polyacrylamide gel and electro transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, United States). Membranes were blocked with 5% (w/v) BSA for 2 h at room temperature and then incubated with primary antibodies with light shaking overnight at 4°C. Primary antibodies against UCP1, PGC-1α, TFAM, NRF1, CREB, P-CREB and GAPDH (abcam) were diluted to a ratio of 1:1,000 in TBST buffer. The membranes were washed 3 times for 5 min each with 10 mL of TBST [10 mM Tris-HCl, 150 mM NaCl and 0.1% (v/v) Tween-20] and then incubated with secondary antibody at room temperature for 2 h. Secondary antibodies goat anti-rabbit or goat anti-mouse (Proteintech, United States) were diluted to a ratio of 1:5,000 in TBST buffer. The membrane was incubated in Western ECL substrate (Thermo fisher or Proteintech, United States) and exposed to Tanon imager, using ImageJ software for image analyses.