Gel imaging system

Total proteins from cells or exosomes were isolated using RIPA lysis buffer (R0020; Solarbio, Beijing, China) and quantified using a BCA detection kit (P0009; Beyotime, Beijing, China) according to the manufacturer’s instructions. Proteins with suitable concentrations were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (WGPVDF22; Servicebio, Wuhai, China). The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-CD63 (ab134045; Abcam, Shanghai, China), anti-TSG101 (ab30871; Abcam, Shanghai, China), anti-VDR (PA5-109276; Invitrogen, Waltham, MA, USA), and anti-GAPDH (GB11002; Servicebio, Wuhai, China), after blocking with 5% blocking solution. Subsequently, the membranes were incubated with a secondary antibody (IH-0011; DingGuo, Beijing, China) at room temperature for 2 h. Protein bands and gray values were visualized and quantified using a gel imaging system (GE Healthcare, Chicago, IL, USA) and Image J software (NIH, Bethesda, MD, USA), respectively.

Total protein was extracted from the liver using a protein extraction kit (Jiancheng Bioengineering Institute, Nanjing, China). Equal amounts of protein per sample were resolved by 10% SDS-PAGE and transferred to Polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skimmed milk in Tris Buffered Saline with Tween-20 (TBST) for 1 h at room temperature, the proteins were incubated overnight with primary antibodies against AMPK (#2532S, Cell Signaling Technology, Danvers, MA, USA), p-AMPK (#2535S, CST), CPT-1A (15184-1-AP, Proteintech Group, Rosemont, USA), FAS (Abp51334, Abbkine, CA, USA), GSK-3β (#9315, CST), PGC-1α (2178S, CST), IL-6 (bs-0379R, Bioss, Beijing, China), TNFα (ab6671, Abcam, Cambridge, UK), and β-actin (Sigma-Aldrich, St Louis, MO, USA) at 4°C. The membranes were then probed with anti-rabbit secondary antibody IgG (HRP) (ab6721, Abcam) or anti-mouse secondary antibody IgG (HRP) (ab197767, Abcam) for 1 h at room temperature. After washing thrice with TBST, the blots were developed using a chemiluminescence reagent (P0018A, Shanghai Beyotime Biotechnology Co., Ltd, China), and the positive bands were visualized with a Gel Imaging System (General Electric, Fairfield, CT, USA). The band intensities were measured using the ImageJ software.

E. coli BL21 (DE3) with pET28a-arcA⁺ was grown in 200 ml of LB medium for 5 h at 30 °C, and protein expression was induced by adding 0.1 mM isopropyl beta-D-1-thio-galactopyranoside (IPTG). The ArcA-His₆ fusion protein was purified using an Ni-NTA-Sefinose Column (Sangon Biotech, Shanghai, China #C600791) in accordance with the protocol provided by the manufacturer. Phosphorylation reactions of ArcA were carried out as described previously [20 (link)]. EMSAs were performed by adding increasing amounts of purified and phosphorylated ArcA-His₆ fusion protein (0, 0.4, 0.8, 1.2,1.6 and 2.0 μg) to the DNA probe (50 ng) in binding buffer (100 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 2 mM DTT, 100 mM KCl, 10% glycerol) for 30 min at 37 °C. DNA–protein complexes were separated by 6% PAGE in 0.5 × TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.0) at 160 V for 1 h. Gels were stained with GelRed for 10 min and imaged using a gel imaging system (GE Healthcare, Chicago, IL, USA).