A2445

Based on the method described in our laboratory [31 (link)], we used the western blot to detect mTOR, p-mTOR, S6, p-S6, Nrf2, and Keap1 protein expression levels. Briefly, the liver was prepared with the RIPA buffer (Thermo Fisher Scientific). Then, the proteins (40 μg from each sample) were separated on 8% or 12% SDS–polyacrylamide gel, depending on the molecular weight of proteins. They were transferred to the PVDF membranes (Thermo Fisher Scientific), blocked with 8% (w/v) skimmed milk in the TBST buffer (20 mM Tris–HCl, 0.1% Tween 20, 150 mM sodium chloride, pH 7.5) for 1 h, washed thrice with the TBST buffer for 10 min each, and followed by the incubation with specific primary antibodies, such as rabbit anti-p-mTOR-S2448 (1 : 2000, AP0094; Abclonal, Wuhan, China), anti-mTOR (1 : 1000, A2445; Abclonal), anti-p-S6- S235/236 (1 : 1000, AP0538; Abclonal), anti-S6 (1 : 1000, A6058; Abclonal), anti-Nrf2 (1 : 1000, A0674; Abclonal), and anti-Keap1 (1 : 5000, A17061; Abclonal) for overnight at 4°C. They were then incubated with goat anti-rabbit secondary antibody (1 : 10000). Immunoreactive bands were visualized via the enhanced chemiluminescence (Cell Signaling Technology) and quantified via the densitometry using ImageJ software (version 1.42, NIH).

Cell lysates were prepared in RIPA lysis buffer, including protease inhibitors (CoWin Biosciences, Beijing, China) and phosphatase inhibitors (CoWin Biosciences, Beijing, China). Equal amounts of proteins were subjected to SDS-polyacrylamide gels. After that, the proteins were transferred to nitrocellulose membranes, and the membranes were blocked with 5% non-fat dried milk for 1 h at room temperature. Each membrane was then incubated with the primary antibody, anti-tubulin (1:1000, AbClonal, AC021), anti-cyclin D1 (1:1000, CST, CST2978), anti-PCNA (1:1000, CST, CST13110), anti-p-eIF2a (1:1000, Abclonal, AP0692), anti-Chop (1:1000, Abclonal, A0221), anti-ATF6 (1:1000, Abclonal, A0202), anti-AKT (1:1000, CST, CST4060), anti-p-AKT (1:1000, CST, CST4691), anti-mTOR (1:1000, Abclonal, A2445), anti-p-mTOR (1:1000, Abclonal, AP0115), anti-P70S6K (1:1000, Abclonal, A2190) or anti-p-P70S6K (1:1000, Abclonal, AP0478), overnight at 4 °C. After incubation with IRDye 800cw or 680cw conjugated antibodies (1:5000 dilution) for 1 h, the membranes were imaged with Odyssey^® CLx Infrared Imaging System. The WB had at least 3 biological replicates.

Protein samples were separated by 10% or 12% SDS-PAGE (SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis) gels and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% fat-free milk in TBST for 2 h, the membranes were incubated with primary antibodies against SLC38A9 (1:1000, ab81687, Abcam, Cambridge, MA, USA), SLC36A1(1:500, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:500, A2445, Abclonal Technology, Wuhan, China), phospho-mTOR (1:500, 2971, Cell Signaling Technology, Danvers, MA, USA), S6K (1:500, sc-230, Santa Cruz Biotechnology, Dallas, TX, USA), phospho-S6K (1:1000, AF3228, Affinity Biosciences, Cincinnati, OH, USA), HA-Tag (1:500, 3724, Cell Signaling Technology, Danvers, MA, USA), and β-Actin (1:1000, BM0627, Boster Biological Technology, Wuhan, China) overnight at 4 °C. The results were visualized using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and enhanced chemiluminescence.