WB uncropped blots & raw raw densitometry

BCAA promotes protein synthesis by activating the Akt/mTORC1, which subsequently regulates its two major downstream effectors, ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Activated S6K1 subsequently inhibits eEF2K, preventing the inactivating phosphorylation of eEF2 and thus promoting translation elongation. Therefore, to explore the underlying signaling mechanism of the C2C12 myotube response to BCAA stimulation, we measured the protein levels of Akt, mTOR, p70S6K, 4E-BP1, and eEF2 by WB after 45 minutes of BCAA treatment. Moreover, the effects of BCAA on the phosphorylation of AMPK, Akt, p70S6K, and eEF2 after 24 hours of treatment were further investigated by conducting WB. Method Total protein was extracted with RIPA buffer from C2C12 myotubes and was then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to polyvinylidene difluoride membranes, which were blocked with 5% non-fat milk dissolved in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies. Finally, membranes were incubated for 1 hour with the second antibodies, and visualized using an enhanced chemiluminescence system. Antibodies used in this study are listed in Table 2. Mean grey value and Integrated Density of each band were measured using ImageJ with Fiji (version 1.35). WB was performed twice for each sample, and statistical analysis was performed on data from three independent experiments (N = 3, n = 2).