Sc 13067

Total protein was extracted from adipose tissues or cultured cells, using RIPA lysis buffer with protease inhibitors cocktail (Selleck). Protein lysates were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were then incubated with primary antibodies, including anti-UCP-1 (Abcam, ab23841), anti-perilipin (Abcam, ab61682), anti-APN (R&D, AF1119), anti-PPARγ (Santa Cruz, sc-7273), anti-PGC1α (Santa Cruz, sc-13067), and β-actin for normalization. Subsequently, incubation with HRP-conjugated antibodies, and then protein signals were detected with electrochemiluminescence (ECL) detection reagents.

For protein isolation, the medium of cells was aspirated and the plates were stored on ice. The cell monolayer was washed gently one time with ice-cold PBS. Excess PBS was aspirated. A volume of 50 µl of RIPA lysis buffer with inhibitors was added to each well of the 24-well plate. RIPA buffer was composed of 50 mM Tris-HCL pH 7.5, 150 mM NaCL 1 mM EDTA, 0.1% sodium deoxycholate, 1% NP-40, 1× protease inhibitor and 1× phosphatase inhibitor. An ice-cold cell scraper was used to scrape the cells. The lysate was transferred to 1.5 ml Eppendorf tubes. The samples were snap-frozen in liquid nitrogen and thawed on ice for three repeated cycles. After 10 min of centrifugation at 12,000× g and 4 °C the supernatant was transferred to fresh tubes and stored on −80 °C. Protein concentration was determined using the bicinchonoinic acid method. Samples were separated by SDS-PAGE after being mixed with 4× Laemmli Sample Buffer containing 10% β-mercaptoethanol and heated to 95 °C for 5 min. Afterwards, proteins were transferred to nitrocellulose membrane for incubation with primary antibodies raised against PGC-1A (sc-13067, Santa Cruz 1:500), UCP1 (sc-6528, Santa Cruz, 1:500). Calnexin (208–880, Calbiochem, 1:5000) served as loading control. Uncropped scans of the blots can be found as a Supplementary Figure in the Supplementary Information.

Quantification of nuclear respiratory factor 1 (NRF1), nuclear factor E2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) was carried out using the Bio-Dot Microfiltration manifold system (Bio-Rad Laboratories). Proteins were extracted from MNCs using Extraction Buffer (ab193970; Abcam), transferred to nitrocellulose membrane using gravity filtration, and blocked using 5% bovine serum albumin, before incubation with antibodies to beta-actin (1:5000; ab8227; Abcam), NRF1 (1:10,000; ab175932; Abcam), Nrf2 (1:3,000; sc-722; Santa Cruz Biotechnology, Santa Cruz, CA) and PGC-1α (1:3,000; sc-13067; Santa Cruz Biotechnology). Immunoreactivity was detected using a horseradish peroxidase–conjugated goat anti-rabbit IgG (1:3,000; ab6721; Abcam) secondary antibody. Protein expression was visualised using ECL Prime Western Blotting Detection reagent (GE Healthcare) in conjunction with a Bio-Rad Universal III Bioplex imager. Densitometric analysis of protein expression was performed using Image Lab software (Image Lab™ version 6.0.1 [2017], Bio-Rad laboratories). Relative expression of NRF1, Nrf2 and PGC-1α was calculated after normalisation to beta-actin levels within the same sample.

The cells were homogenized in RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors to extract proteins. The protein samples were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. Then, the membranes were incubated with the primary antibodies. Anti-PGC-1α (sc13067, Santa Cruz, 1:1000), anti-EZH2 (ab3748, Abcam, 1:1000), and anti-β-actin (A5441, Sigma-Aldrich, 1:2500) antibodies. The protein bands were analyzed using Image Lab software (Bio-Rad), and the gray values of experimental proteins were detected using ImageJ software.