Nuclear or cytoplasmic protein extraction kit

Proteins expression was measured by western blotting as described previously (28 (link)). Nuclear and cytoplasmic proteins were extracted from cultured cells using a nuclear or cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology), respectively (Cells were seeded into plates at a density of 3.5 × 10⁴/ml). Protein concentrations were measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). Then, cell lysates (10 µg/lane) were separated by SDS-PAGE (10%) and electrophoretically transferred onto a polyvinylidene fluoride membrane. The membrane was then washed with TBS with Tween-20 (TBST) three times, and blocked with 5% non-fat milk in TBST (1% Tween-20) for 2 h at room temperature. The membrane was incubated with Nrf2 (1:1,000), keap1 (1:300), p65 (1:3,000), IκBα (1:1,000), GAPDH (1:1,000) and Lamin B (1:1,000) antibodies overnight at 4°C, and incubated with HRP-coupled secondary antibodies (1:2,000) for 1 h at room temperature. Proteins were detected using enhanced chemiluminescence (Fdbio Science). The protein intensity was measured using Image J software (version 1.48; National Institutes of Health). The relative protein levels were normalized to GAPDH/Lamin B1 protein.