Anti cpt1α

Total proteins were isolated from white adipose tissues using RIPA (Solarbio Life Science, Beijing, China) buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a protein assay reagent (Beyotime Biotechnology, China) and equal amounts of protein (60 μg) were loaded in each well of an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. After the proteins were transferred onto a polyvinylidene difluoride membranes, the blots were blocked with 5% bovine serum albumin, followed by incubation overnight at 4°C with the following primary antibodies: anti-AMPKα (23A3), anti-phospho-AMPKα (Thr172), anti-ACC, anti-FAS, and anti-CPT1α (Cell Signaling Technology, United States). After three washes with TBST buffer, the blots were incubated with appropriately diluted horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immunoblots were visualized with an enhanced chemiluminescence detection kit (Tiangen Biotech, Beijing, China). Protein levels were normalized to tubulin as a loading control.

Chemiluminescent based on horseradish peroxidase (HRP) were used to detect and visualize western blots. The following antibodies were used: anti-CPT1α (12252S, Cell Signaling Technology, Beverly, MA, USA), PACS2 (GTX17244, GeneTex, Irvine, CA, USA), VE-cadherin (ab33168, Abcam), β‐actin (A5441, Sigma‐Aldrich), AKT (2938S, Cell Signaling Technology), p-AKT (9018S, Cell Signaling Technology), Smad2 (5339T, Cell Signaling Technology), p-Smad2 (ab280888, Abcam, UK), PPARα (ab215270, Abcam, UK), and HRP conjugated goat anti-rabbit IgG secondary antibody (AS09 602, Agrisera, Vannas, Sweden).

Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco (Rockville, MD, USA). Penicillin-streptomycin solution was purchased from Hyclone (Provo, UT, USA). Insulin, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3T3-L1 preadipocytes were purchased from ATCC (Manassas, VA, USA). Bilobalide was obtained from the National Institutes for Food and Drug Control (Beijing, China). Primary antibodies specific for anti-pACC1 (S79), anti-ACC1, anti-FASN, anti-perilipin A, anti-ATGL, anti-C/EBPα, anti-PPARγ, anti-HSL, anti-pHSL (S563), anti-GLUT-4, anti-CPT-1α, anti-AMPK, and anti-pAMPK (T172) were acquired from Cell Signaling Technology (Danvers, MD, USA). Anti-SREBP-1c was acquired from Abcam (Cambridge, UK). Anti-β-Actin and goat anti-mouse and goat anti-rabbit IgG secondary antibodies were obtained from Boster Biological Technology (Pleasanton, CA, USA). The AMPK assay kit was purchased from Cell Signaling Technology.