β3 adrenergic receptor

Membrane proteins or total cell proteins were extracted from homogenized LV tissue. The primary antibodies included NOX2, NOX4, p22^phox, p47^phox, p67^phox, Rac-1 (1 : 500, Abcam); eNOS, p-eNOS^Ser1177, p-eNOS^Thr495, p-eNOS^Ser114, iNOS, nNOS (1 : 1000, Cell Signaling Technology), β3-adrenergic receptor (1 : 500, Abcam), and β-actin (1 : 5000, Abcam).

Inguinal white adipose tissue was harvested using the Adipose Protein Extraction Kit (MinuteTM) for protein extraction. 80 μg of adipose tissue was homogenized with 300 μl extraction buffer. After centrifuging at 2,000 rpm for 1 min, the tissue was transferred to a filter cartridge with a collection tube and incubated at −20°C for 15–20 min. After incubation, it was immediately centrifuged at 2000 rpm for 1–2 min with cap open. The flow-through contained total proteins from adipose tissue. Then, 20 μg of total tissue lysate was separated by SDS-PAGE (10% of separation gel and 5% of concentration gel). The electrophoresis contained 80 V for 0.5 h and 120 V for 1 h. The protein bands were transferred to a PVDF membrane using Trans-Blot (Bio-Rad), and 5% BSA was added for 2 h for blocking the membrane. The membrane was then reacted with primary antibodies against: UCP1 (1:1000, Abcam), norepinephrine transporter (1:1000, Abcam), β-actin (1:1000, Abcam), β3 adrenergic receptor (1:1000, Abcam), and tyrosine hydroxylase (1:1000, Abcam) under 4°C overnight. Incubation with the corresponding secondary antibodies (diluted to 1:3000, Abcam) was performed at room temperature for 1 h.