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).支链氨基酸（BCAA）可通过激活Akt/雷帕霉素靶蛋白复合物1（mTORC1）通路促进蛋白质合成，继而调控其两大下游效应因子——核糖体蛋白S6激酶1（ribosomal protein S6 kinase 1，S6K1）与真核起始因子4E结合蛋白1（eukaryotic initiation factor 4E binding protein 1，4EBP1）。活化的S6K1可进一步抑制eEF2激酶（eEF2K），阻止eEF2发生失活磷酸化，从而促进翻译延伸过程。为探究C2C12肌管对BCAA刺激的潜在信号转导机制，本研究在BCAA处理45分钟后，通过蛋白质免疫印迹（WB，Western Blot）检测了Akt、mTOR、p70S6K、4EBP1及eEF2的蛋白表达水平。此外，本研究还进一步开展了蛋白质免疫印迹实验，以分析BCAA处理24小时后对AMPK、Akt、p70S6K及eEF2磷酸化水平的影响。 实验方法 总蛋白采用RIPA缓冲液从C2C12肌管中提取，随后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE）进行分离。将分离得到的蛋白转移至聚偏氟乙烯膜，使用含0.05%吐温20的Tris缓冲盐溶液（TBS-T）配制的5%脱脂牛奶于室温封闭1小时。之后将膜与一抗于4℃条件下孵育过夜。最后将膜与二抗孵育1小时，通过增强化学发光系统完成显影成像。本研究使用的抗体详见表2。采用搭载Fiji插件的ImageJ软件（版本1.35）测定每条条带的平均灰度值与积分光密度。每个样本重复两次蛋白质免疫印迹实验，所有数据均来自3次独立重复实验（N=3，n=2）并进行统计分析。