Trans blot turbo transfert system

Total proteins were extracted in RIPA lysis buffer (Sigma) containing 1% protease inhibitor cocktail (Sigma) and 10% phosphatase inhibitor (Roche). Proteins were quantified using the Pierce BCA Protein assay kit (Thermo Fisher Scientific) and loaded using NuPAGE™ 3%–8% and Tris-acetate (Invitrogen). Total proteins were then transferred to PVDF membrane (Bio-Rad) with a Trans-Blot Turbo Transfert system (Bio-Rad), blocked with Odyssey blocking buffer (OBB) containing 0.1% Tween-20, and incubated overnight with the primary anti-neurofibromin antibody (abcam, ab17963, 1/1000) diluted in the OBB containing 0.1% Tween-20. The membrane was then incubated for 1 h with the corresponding IRDye secondary antibodies (LI-COR). Immunoreactive protein bands were revealed using an Odyssey CLx Imager (LI-COR) according to the manufacturer’s protocol. Anti-beta-Actin antibody (Santa Cruz Biotechnologies, sc4778, 1/10,000) or anti-vinculin (abcam, ab129002, 1/1000) was used to verify equal protein loading.

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).