P nrf2

EGCG (purity ≥ 98%) was provided by Teaturn Bio-pharmaceutical Co., Ltd. (Wuxi, China). Anti-TFR1, FPN, FTH/L, NRF2, pNRF2, GPX4, P62, and HO-1 were purchased from Abcam; anti-KEAP1, SLC3A2, and SLC7A11 were purchased from Cell Signaling Technology; and β-actin was purchased from Santa Cruz biotechnology.

Samples were incubated for 10 minutes at 95°C in the loading buffer. Samples were then subjected to electrophoresis on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies of Bb (1:500; Quidel catalog A227), GRP78 (1:1,000; Proteintech catalog 11587-1-AP), CHOP (1:500; Proteintech catalog 15204-1-AP), p-Nrf2 (1:500; Abcam catalog ab76026), cleaved caspase-3 (1:1,000; Cell Signaling Technology catalog 9661), NF-κB p65 (1:1,000; Cell Signaling Technology catalog 8242), p-p65 (1:1,000; Cell Signaling Technology catalog 3033), IκBα (1:1,000; Cell Signaling Technology catalog 4814), p-IκBα (1:1,000; Cell Signaling Technology catalog 9246), IKBKB (1:500; Proteintech catalog 15649-1-AP), histone H3 (1:1,000; Proteintech catalog 17168-1-AP), and β-tubulin (1:2,000; Cell Signaling Technology catalog 2128), followed by horseradish peroxidase–conjugated secondary antibodies (Proteintech catalog SA00001-1 and SA00001-2). Proteins were visualized on autoradiographic film using an ECL Plus Western blot detection system (GE Healthcare, now Cytiva).

The formalin-fixed, paraffin-embedded liver tissues were sectioned at 5 μm. Then, the sections were dewaxed in xylene and rehydrated through a graded series of alcohol to water. The liver slices were first treated for 60 min with primary antibodies against Iκ-Bα, NF-κB, Nrf2, p-Nrf2, and HO-1 (obtained from Abcam, Cambridge, UK) and then incubated for 120 min with biotinylated anti-mouse secondary antibodies (obtained from Abcam, Cambridge, England). Finally, the tissue sections were then incubated with streptavidin-horseradish peroxidase (Abcam, Cambridge, UK), and a light microscope was used to measure the brown shade under 3,3’-diaminobenzidine tetrahydrochloride [18 (link)]. Three randomly selected areas were observed per slide, and the mean density was calculated using ImageJ software.

Total proteins were extracted using ice-cold RIPA buffer containing protease inhibitor cocktail (Roche). Protein extraction kit (Beyotime) was used to extract nuclear and cytoplasmic protein. The concentrations were determined using a BCA kit (Beyotime). Equal amount of protein from each sample was run in 8–12% Tris-glycine SDS-PAGE gel, followed by transfer to PVDF membrane (Millipore). Membranes were blocked with 5% skim milk and probed with primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated anti-IgG (Beyotime) for 1 h at room temperature. The intensity of bands was visualized and determined using an enhanced chemiluminescence detection system (Bio-Rad Laboratories). Primary antibodies used were: γ-H2AX, GPX-1, Akt, p-Akt, p21, S6, p-S6, p-LKB1, LC3B, AMPK, p-AMPK, Atg7, p62, ACC, p-ACC (1:1000, Cell Signaling), SOD1, SOD2, Catalase, p-NRF2, FOXO3a, p-FOXO3a (1:1000, Abcam), p16, NRF2, Lamin B1, GCLC, GCLM, HMOX1, NQO1, TXN, Keap1, SIRT1, SIRT3, SIRT6, LKB1, CAMKK2(1:1000, ProteinTech), Actin and GAPDH (1:1000, Beyotime). The primary images (Supplementary Fig. 18) were cropped for presentation.