Nrf2 primary antibody

Cytoplasmic and nuclear proteins in the liver and muscle were extracted using nuclear and cytoplasmic protein extraction kits (Beyotime, Shanghai, China). Proteins were separated by SDS-PAGE (10% gel), transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes (Biosharp). After blocking with 5% skim milk powder (Biosharp) in TBST (0.1% TWEEN-20), the membranes were incubated with Nrf2 primary antibody (Proteintech, Chicago, IL, USA) overnight, washed with TBST, and incubated with secondary antibody at room temperature for one hour. Chemiluminescent Substrate System from KPL was utilized for final detection.

Nuclear and cytoplasmic protein extracts were harvested from HepG2 cells treated with or without PME (100 μg/mL) for 6 h and were prepared as described [12 ]. Protein samples (20 μg) were separated using electrophoresis on a 4%–20% gradient gel prepared by a TOOLS HR Gradient gel solution (TOOLS Biotech, New Taipei City, Taiwan) and transferred to a polyvinylidene difluoride transfer membrane (Immunobilon-P, Millipore, Bedford, MA). Subsequently, the membrane was blocked using 2% bovine serum albumin and then incubated with an Nrf2 primary antibody (1:1000, ProteinTech, Chicago, IL) or lamin β1 (1:1000, Abcam, Cambridge, MA) and α-tubulin (1:1000 GeneTex, Irvine, CA) at 4°C overnight. After incubating with a secondary antibody, the Immobilon™ Western Chemiluminescent HRP Substrate (Merck-Millipore) was used to detect reactive protein signals.