Horseradish peroxidase conjugated polyclonal goat anti rabbit secondary antibody

C2C12 cells for western blot were scraped in cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Rockford, IL) containing protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein concentrations were measured by the bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). Samples (one per experiment) were denaturated in SDS–PAGE sample buffer for 5 min at 90°C, loaded onto a SDS PAGE gel and transferred to a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). After transfer, the membrane was blocked overnight at 4°C with 2% ECL Advance Blocking Agent (GE Healthcare) in TBS with 0.01% Tween 20 (Sigma–Aldrich). Subsequently, the membrane was washed and incubated for 1 h at room temperature with primary antibody against p‐Akt (Ser473; 1:4000), total Akt (1:4000), p‐S6 (Ser235/236; 1:1000), total S6 (1:1000), and β‐tubulin (1:2000) (all Cell Signaling Technology). After washing, the membrane was incubated for 1 h at room temperature with horseradish peroxidase‐conjugated polyclonal goat anti‐rabbit secondary antibody (1:4000; DakoCytomation, Glostrup, Denmark) and the membrane was analyzed with the enhanced chemiluminescence method (ECL Advance; GE Healthcare). Western blots were quantified using ImageJ. Total Akt, p‐Akt, total S6, and p‐S6 were normalized to β‐tubulin.