Ncl dys1 primary monoclonal antibody

Protein extracts were obtained from dissected brain structures (cortex, hippocampus, cerebellum) treated with RIPA lysis and extraction buffer (Thermo Fisher Scientific) complemented with SDS powder (5% final; Bio‐Rad, Marnes‐la‐Coquette, France), and the total protein concentration was determined with the BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were denatured at 100°C for 3 minutes, and 25 μg of protein were loaded onto NuPAGE 3–8% Tris‐Acetate Protein gels (Invitrogen), following the manufacturer's instructions. Dystrophin protein was detected by probing the membrane with NCL‐DYS1 primary monoclonal antibody (NCL‐DYS1; Novocastra, Newcastle, UK), and vinculin was detected as the internal control with the hVin‐1 primary antibody (Sigma, St Louis, MO, USA), followed by incubation with a goat anti‐mouse secondary antibody (IRDye 800CW Goat anti‐mouse IgG; Li‐Cor, Germany). Bands were visualized using the Odyssey CLx system (Li‐Cor, Lincoln, NE, USA), and quantification was carried out using the Empiria Studio software (Li‐Cor) based on a standard curve specific of each brain structure and made of a mix of WT and mdx control lysates to obtain defined percentages of dystrophin (0, 5, 10, 15%, or 0, 10, 15, 30% of corresponding WT tissues).