Chemiluminescence imaging system

For isolation of total proteins, cells were rinsed with cold PBS and resuspended in lysis buffer (0.5 M NaCl, 50 mM Tris, 10% glycerol, 1.0 mM EDTA, 1mM BME, 1% NP40, plus proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). The supernatant was collected by centrifugation at 12,000 g for 15 min at 4 °C. The concentration of isolated protein was determined by the Bradford method. Isolated proteins (40 μg) were loaded and analyzed by immunoblotting. Membranes with transferred protein were incubated with primary antibodies in the blocking buffer (5% non-fat dry milk, 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20 on an orbital shaker at 4 °C overnight. Membranes then were incubated with horseradish peroxidase-linked secondary antibodies (anti-rabbit, anti-mouse, or anti-goat IgG). Antibody-bound protein bands were detected using high sensitive Immoblot Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA), and photographed with Bio-Rad Chemiluminescence Imaging System (Bio-Rad Laboratories, Inc. Hercules, CA, USA)

Western blot analysis was performed using standard procedures. Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China). The protein was prepared and quantified by Bradford analysis (Beyotime, China). The same amounts of protein were extracted by 10% and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Germany). Membranes were blocked with 5% milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour and incubated with primary antibody for 2 hours at RT or overnight at 4°C. The membranes were washed 3 times with PBST and then incubated with species-specific secondary antibodies for 1.5 hours at RT. Next, the membranes were washed 3 times with TBST, once with tris-buffered saline (TBS), and then imaged using the Bio-Rad chemiluminescence imaging system (Bio-Rad Laboratories, Inc., USA). All image quantifications were performed using Fiji ImageJ software.
The following primary antibodies were used: rabbit polyclonal antibody against β-catenin (Proteintech Group, China), rabbit polyclonal antibody against GSK3β (Beyotime, China), and mouse polyclonal antibody against β-actin (Beyotime, China). The following secondary antibodies were used: goat anti-mouse IgG/AP (Beyotime, China) and goat anti-rabbit IgG/HRP (Beyotime, USA).

Total protein and nuclear protein were isolated from the liver tissues by RIPA buffer (Beyotime, Shanghai, China) and Nuclear Extraction Kit (Invent, Erlangen, Germany), respectively. The protein concentration was determined by the BCA method (Beyotime, Shanghai, China). Proteins were separated using 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene fluoride (PVDF) membranes (Merck Millipore Ltd., Darmstadt, Germany). Next, the transferred membranes were blocked using Protein Free Rapid Blocking Buffer (EpiZyme Biotechnology, Shanghai, China). Membranes were incubated with primary specific antibodies at 4 °C overnight. Primary specific antibodies included anti-SREBP-1c, anti-PI3K, anti-p-PI3K, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, and anti-β-actin. Then, the PVDF membranes were washed and incubated with goat anti-mouse IgG (H+L) HRP conjugate or goat anti-rabbit IgG (H+L) HRP conjugate at room temperature for 1 h. Finally, Luminata Forte Western HRP substrate (Millipore, Darmstadt, Germany) was added to detect a specific protein expression using the Bio-Rad chemiluminescence imaging system (Bio-Rad, CA, USA). The intensity of the bands was quantitated by ImageJ (Rawak Software Inc., Stuttgart, Germany).

Aluminium trichloride, urethane, hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA), maltolate (Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-CREB (Ser133) (Cell Signalling, USA), anti-phospho-tau (Ser396), anti-PKA (Affinity, USA), anti-PRKACB (Merck, USA), anti-tau (Sercivebio, CN), anti-GAPDH mouse, goat anti-rabbit immunoglobulin (IgG), goat anti-mouse IgG, miRNA extraction kit, high sensitivity chemiluminescence liquid ECL, protein quantitative kit, tissue protein extraction kit, lane marker loading buffer (Reducing, 5x), EasyQuick RT MasterMix, Acry (Beijing ComWin Biotech Co., Ltd. CN), all-in-one miRNA qRT-PCR detection kit, primer for miR-200a-3p, primer for U6 (FulenGen, CN), DYC Z-24DN electrophoresis tank DYCP-40C semi-dry membrane transfer instrument (Liuyi instrument factory, Beijing), Bio-Rad chemiluminescence imaging system (Bio-Rad, USA), and ethyl carbamate (Sigma-Aldrich, St. Louis, MO, USA) were used in this study.

The proteins in tissues were lysed using lysis buffer, and same amount of samples was separated by 8% SDS-PAGE. After electrophoresis, the protein was transferred to nitrocellulose membrane (Millipore, USA). After blocking with TBST, the membranes were incubated with primary antibody (1:1000) at 4° C overnight. After washing twice with PBS, the membranes were incubated with secondary antibody (1:2000) for 2 h at room temperature. Membranes were visualized using an enhanced chemiluminescence kit (#34096, Thermo Fisher, Waltham, MA, USA) under the chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). The antibodies used in this study were listed as follows: Mouse monoclonal to GSK-3β (#ab93926, abcam, Cambridge, UK), Rabbit monoclonal to p-GSK-3β (##9323, CST, Danvers, CO, USA,), Mouse monoclonal to HK-2 (#ab209847, abcam, Cambridge, UK), Rabbit monoclonal to SREBP-1c (#ab28481, abcam, Cambridge, UK), Rabbit monoclonal to CPT-1 (#ab234111, abcam, Cambridge, UK), Mouse monoclonal to β-actin (#ab8226, abcam, Cambridge, UK), Goat anti-mouse antibody (#ab205719, abcam, Cambridge, UK), Goat anti-rabbit antibody (#ab205718, abcam, Cambridge, UK). The experiment was performed 3 times independently.