Ab174327

After six weeks of drug therapy, all mice were euthanized by intraperitoneal injection with 0.1% pentobarbital sodium. The adipose tissue from the other three groups of mice were removed and stored at −80°C to examine the protein expressions of Insig-1, SCAP, and SREBP-1c. The protocol for western blotting was previously described by us [17 (link)]. Primary antibodies including Insig-1 (Abcam, ab70784, 1 : 200), SREBP-1c (Abcam, ab28481, 1 : 1000), SCAP (Abcam, ab19013, 1 : 1000), IRS-1 (Abcam, ab52167, 1 : 1000), PI3K (Abcam, ab151549, 1 : 1000), Akt (Abcam, ab8805, 1 : 500), mTORC1(Abcam, ab2732, 1 : 200), PAQR3 (Abcam, ab174327, 1 : 250), and β-actin (Beijing Zhongshan Golden Bridge Biotechnology Co., TA-09, 1 : 1000) were used in this study. Protein expression was detected with an enhanced chemiluminescence detection system (Vigorous, Beijing, China).

ALL samples or cells were lysed through RIPA lysis buffer (Beyotime). Proteins were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then passed onto polyvinylidene difluoride membranes. After being blocked with 5%‐skim milk, membranes were incubated with primary antibodies: anti‐PAQR3 (1:1000; ab174327; Abcam), anti‐Nrf2 (1:1000; ab137550; Abcam) and anti‐β‐actin (1:1000; ab8226; Abcam) overnight at 4°C. Next, membranes were incubated with HRP‐conjugated secondary antibodies. Bands were observed by the ECL chemiluminescent detection system (Thermo Fisher Scientific).