Anti nrf2

E11 cells were lysed directly in RIPA buffer (Thermo Scientific, Lithuania, USA) containing protease inhibitor (Sigma, St. Louis, USA). 20 μg of protein were loaded in each lane of 8~10% SDS—PAGE gels, transferred onto Immobilon-P Transfer Membrane (Millipore, Bedford, USA), probed with antibodies, and analyzed with Las-4000 imaging system (Fujifilm, Valhalla, USA). 200 ug of cell lysates were mixed with antibody and the immunocomplexes were collected on Dynabeads protein A (Life Technologies, Carlsbad, USA). Antibodies are indicated as following: anti-Nrf2, anti-AMPK1/2, anti-β-actin (Cell Signaling, Danvers, USA), control rabbit IgG (Santa Sruz, Dallas, USA) and anti-HO1 (ENZO Biotech, New York, USA).

HRP-conjugated anti-β-ACTIN antibody, Filipin and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich/ Merck Millipore. Alexa488-conjugated anti-rabbit IgG, HRP-conjugated anti-rabbit total IgG and light chain specific IgG antibodies were purchased from Jackson ImmunoResearch, USA; PE-conjugated F4/80 was procured from Tonbo Biosciences, USA. Alexa Fluor 660 conjugated CD68 was purchased from ThermoFischer Scientific. Anti-G9a, anti-SIRT6, anti-H3K9me1, anti-H3K9me2, anti-H3K9Ac, anti-Ser33/37/Thr41 phospho-β-CATENIN, anti-Ser9 phospho-GSK-3β, anti-β-CATENIN, anti-NRF2, anti-HO1 and anti-TRXR1 antibodies were obtained from Cell Signaling Technology, USA. Anti-LRP2 antibody was purchased from Santa Cruz Biotechnology, USA; anti-SREBP2 antibody was procured from Abcam, USA; and anti-NQO1 antibody was purchased from Calbiochem, USA.

Upon 24 h of infection and treatment, A549 cells were collected and lysed in cold RIPA buffer [20 mM Tris–HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% SDS and 1% sodium deoxycholate] supplemented with phenylmethyl-sulphonyl fluoride, protease inhibitor mixture, and phosphatase inhibitor (Sigma Aldrich, St. Louis, MO, USA). After 30 min of incubation, the cell lysates were centrifuged (14,000× g, 30 min, 4 °C). The supernatants obtained from the centrifugation were collected, and the protein concentration was determined by a Bradford protein assay. For the SDS-PAGE electrophoresis procedure, cell lysates samples were prepared by adding sodium dodecyl sulfate (SDS) buffer and DL-dithiothreitol. After the protein transfer, the nitrocellulose membrane was blocked with 10% milk solution (Bio-Rad Laboratories, Berkeley, CA, USA) diluted in T-TBS (0.01% Tween 20 plus Tris-buffered saline) for 1 h at room temperature and then incubated with a polyclonal goat anti-influenza antibody (anti-Flu, AB1074 Merck Millipore, Darmstadt, Germany), anti-Nrf2 (#12721 Cell Signaling) or with anti G6PD (#12263 Cell Signaling) overnight at 4 °C. After three washes with T-TBS, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody and developed using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA). Actin was used as a loading control.

Glycyrrhizin was purchased from the National Institute for Food and Drug Control (Nanjing, China). Sodium iodate was procured from Sigma Industrial Company (Shanghai, China, purity 98%). Anti‐p‐Akt (Ser473), anti‐Akt, anti‐Nrf2, anti‐HO‐1, anti‐cleaved caspase‐3 and anti‐β‐actin were obtained from Cell Signaling Technology (Shanghai, China). Human adult pigment epithelial‐19 (ARPE‐19) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Hepatic expressions of CYP2E1, peroxisome proliferator-activated receptor gamma (PPAR-γ), nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), phosphorylated map kinase kinase 4 (p-MKK4), phosphorylated c-Jun N-terminal kinases (p-JNK), p62, phosphorylated p62 (p-p62), nuclear factor erythroid 2-related factor 2 (NRF2), β-actin were determined by performing western blotting. Similar-weighted liver sections obtained from mice in each group were homogenized in lysis buffer and centrifuged. Proteins were separated by performing electrophoresis on 4–12% gradient Bis-Tris gels; transferred to Hybond ECL nitrocellulose membranes; and immunoblotted using anti-CYP2E1 (Abcam, Cambridge, UK), anti-PPAR-γ (Cell Signaling, Danvers, MA), anti-NF-κB (Cell Signaling), anti-ERK (Cell Signaling), anti-p-ERK T202/Y204 (Cell Signaling), anti-p-MKK4 S257 (Cell Signaling), anti-p-JNK T183/Y185 (Cell Signaling), anti-p62 (Cell Signaling), anti-p-p62 S349 (Cell Signaling), and anti-NRF2 (Cell Signaling) antibodies. Antibody against β-actin (Sigma-Aldrich) was used to assess equal loading.

4-Octyl itaconate was initially supplied by Professor Richard Hartley and results were later confirmed with commercially available 4-OI (Sigma Aldrich). Pam3CSK4, dimethyl fumarate, diethyl maleate, indomethacin and NS-398 (Sigma Aldrich) were also used. Antibodies used were anti-β-actin (Sigma Aldrich), anti-COX2 (Abcam), anti-phospho-cPLA2 (Ser505), anti-cPLA2, anti-NRF2, anti-KEAP1, anti-ATF4, anti-phospho-NF-kB p65 (Ser536), anti-NF-kB p65, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-p38 MAPK, anti-phospho p44/42 MAPK (Erk1/2) (Thr202/Tyr 204) and anti-p44/42 MAPK (Erk1/2) (Cell Signaling). Anti-mouse IgG and anti-rabbit IgG secondary horseradish peroxidase-conjugated antibodies (Jackson Immunoresearch) were also used. A PGE₂ ELISA kit was used (Enzo Life Sciences). The Silencer Select siRNAs against NRF2 (s70522), ATF4 (s62689) and Anxa1 (s69299), as well as the Silencer Select negative control, were used (ThermoFisher Scientific).