Anti heme oxygenase 1 ho 1

Anti-SREBP-1c, anti-phospho-AMPKα, anti-phospho-acetyl-CoA carboxylase (ACC), anti-phospho- liver kinase B1 (LKB1), anti-phospho-YAP, anti-YAP, anti-phospho-large tumor suppressor kinase 1 (LATS1), anti-heme oxygenase 1 (HO-1), anti-Lamin A/C, and anti-β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Nrf2 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Compound C (Com C), verteporfin (VP), T0901317 (T090), Harris hematoxylin and eosin, and Oil Red O solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol (PEG) 400 was obtained from Yakury Pure Chemical Co., Ltd. (Kyoto, Japan). SC is a medicinal-standard herb manufactured at a GMP facility (Nonglim Saengyak, Seoul, Republic of Korea) certified by the Korean FDA and was prepared and standardized as previously described [13 (link)].

Homogenized rat liver was lysed in 200 μl RIPA lysis buffer (Beyotime, P0013B) with 1% phenylmethyl sulfonylfluoride and 4% complete protease inhibitor cocktail mix (Roche, Mannheim, Germany). Extracts were centrifuged at 14,000 g for 15 min at 4°C. Eighty micrograms of total protein were used for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by transferring blotting to nitrocellulose membrane (Millipore Corp., Billerica, MA, USA). Membranes were then blocked with 5% nonfat dried milk in PBS for 1 hr with gentle shaking. Membranes were incubated first with anti‐Janus kinase 2(JAK2), anti‐signal transducer and activator of transcription 3 (STAT3) and their phosphorylated species, anti‐cytochrome P450 2E1(CYP2E1), anti‐Nuclear factor erythroid 2 related factor 2 (Nrf2), anti‐heme oxygenase‐1(HO‐1), anti‐NAD(P)H: quinone oxidoreductase 1 (NQO‐1), and anti‐β‐actin antibody [Cell Signaling Technology] were incubated overnight at 4°C, in 1% BSA in PBS overnight at 4°C with shaking, washed and incubated with secondary antibodies for 2 hr at room temperature. Finally, the samples were visualized by enhanced chemi‐luminescence. After scanning, band density was analyzed using Image J 1.33 software (National Institutes of Health, Bethesda, MD, USA).

Myoblasts were seeded at 1.10⁵ in 35 mm collagen-coated dishes, cultured in proliferation medium for 24 h, pre-incubated or not with onopordopicrin for 24 h and then incubated or not with a sub-lethal concentration (70 μM) of hydrogen peroxide (H₂O₂) for 24 h. Protein extracts (25 µg/well) were separated by SDS-PAGE gel electrophoresis one hour at 25 mA/gel using Mini Protean Precasts Gel 4–15% (Bio-Rad) and transferred in 5 min (1.3 A, 25 V) with Transblot turbo transfert system (Bio-Rad, Schiltigheim, France), to nitrocellulose membranes (Transblot turbo transfert pack 0.2 µm nitrocellulose, Bio-Rad, Schiltigheim, France), blocked at room temperature with Odyssey blocking buffer (Eurobio Scientific, Les Ulis, France) and probed with the rabbit polyclonal anti-heme oxygenase 1 (H-O1) (ref: 43966S; 1/2000; Cell Signalling, Danvers, MA, USA) antibodies, rabbit polyclonal anti phosphorylated γ-H2AX (ref: 9718S; 1/3000; Cell Signalling, Danvers, MA, USA) antibodies and rabbit polyclonal anti Nrf2 (ref: 127215; 1/1000; Cell Signalling, Danvers, MA, USA) antibodies followed by IRDye^® 680RD and IRDye^® 800RD secondary antibodies (Eurobio Scientific, Les Ulis, France). Fluorescence was quantified with the Odyssey software. Data were normalized to α tubulin expression (alpha tubulin mouse antibody (ref: T9026; 1/10,000; Sigma-Aldrich, Lyon, France).