Ecl advance detection kit

Total protein extracts from mice gastrocnemius and tibialis anterior muscles were obtained in homogenization buffer (15 mg of dry tissue/150 μl of 20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Triton X-100, 10 mM EDTA) added with protease inhibitor cocktail (Sigma Aldrich). After sonication and centrifugation at 14000 × g (20 min at 4°C) supernatants were collected and assayed for protein content by Bradford detection kit (Bio-Rad Laboratories, Hercules, United States). Protein separation and analysis (15 μg/well) was performed by Mini-PROTEAN^® TGX^TM Gels (BioRad, United States) and by transfer onto nitrocellulose membranes. After saturation with 5% non-fat dry milk (1 h at room temperature), membranes were probed with gp91phox antibody (1:1000, BD Transduction Laboratories, United States) in 5% non-fat dry milk overnight at 4°C, and incubated with HRP-conjugated secondary antibody (mouse 1:5000, Jackson Immunoresearch) for 1 h at room temperature. Detections were performed on X-ray film (Aurogene, United States), using ECL Advance detection kit (Amersham Biosciences, United States) and signal intensity visualized by Kodak Image Station and analyzed by ImageJ software (NIH, United States). Values were normalized with mouse anti-GAPDH (1:2500, Sigma-Aldrich, Italy).

Cells were lysed in RIPA lysis buffer, including protease inhibitors, according to the manufacturer’s instructions (Santa Cruz Biotechnology, Dallas, TX, USA). Protein concentrations were determined using the Bradford method (Bio-Rad). A total of 20 μg protein lysates were separated on 4–12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Bio-Rad). For detection of BRAF^V600E, HMOX-1, phosphorylated AKT (B-12 sc-377556), phosphoERK (Cell Signaling #4370), GAPDH (G-9 sc-365062), and actin (C-2: sc-8432)-specific antibodies were used at 1:1000 dilution, and protein signals were developed by an ECL advance detection kit according to the manufacturer’s instructions (Amersham Bioscience, Piscataway, NJ, USA). Uncropped Western Blot images could be found at Original blots and intensity ratio.