Nuclear extraction kit

Nuclear extracts were prepared from lung tissues using a nuclear extraction kit (Invent Biotechnologies, Inc., Plymouth City, MN), according to the manufacturer’s instructions. The total cellular extracts were prepared from BAL-recovered cells using a mammalian cell lysis reagent (Merck), according to the manufacturer’s instructions. Ten to twenty micrograms of nuclear extracts was separated with 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After the blocking of nonspecific sites, the PVDF membranes were incubated with anti-Nrf2 antibodies (H-300) or anti-Nramp1 antibodies (E-2), followed by incubation with horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized by image scanning using a LAS-4000 Imager (GE Healthcare). Lamin B or β-actin was used as an internal control. Values were normalized to lamin B to evaluate expression of Nrf2. Values were normalized to β-actin to evaluate expression of Nramp1.

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).