Bca protein quantitation kit

The protein was extracted by a Plant Total Protein extraction kit (Bestbio, Shanghai, China). Ginseng hairy roots were ground with the cryogenic grinding machine and kept at 4 °C. Total protein was determined using the BCA Protein quantitation kit (Bestbio).

The antibodies used were MyHC (381620, ZENBIO), Myomaker (A18158, ABclonal), HuR (382170, ZENBIO), β-tubulin (200608, ZENBIO), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (511203, ZENBIO). Total protein from in vitro cultured MuSCs was extracted using a total protein extraction kit (Solarbio, Beijing, China) and quantified using the BCA Protein Quantitation Kit (BestBio, Shanghai, China). In brief, ~20 µg of the qualified protein per samples were separated on a 10% SAS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), blocked with 5% non-fat milk for 2 h at 37 °C, incubated with primary anti-rabbit for MyHC (1:500), Myomaker (1:1000), and HuR (1:1000) at 4 °C overnight and with a secondary antibody conjugated with HRP (1:10,000) for 1.5 h at 37 °C. Eventually, protein bands were exposed via the enhanced chemiluminescence detection system (BeyoECL Plus, TIANGEN, Beijing, China). β-tubulin antibody (1:1000) worked as a loading control.

To measure the protein level of MyoD, the total proteins from cultured SMSCs were extracted using the Total Protein Extraction Kit (BestBio, Shanghai, China) and quantified by employing BCA Protein Quantitation Kit (BestBio, Shanghai, China) according to the manufacturer’s instructions. In brief, with ~20 μg of protein per sample, Western blotting was performed by separating protein on a 12% SAS-PAGE, transferring to a PVDF (Millipore, Burlington, MA, USA), blocking with 5% milk in TBS, and then incubating the membrane sequentially with the primary anti-mouse MyoD (1:1000) (Abcam, Bristol, UK) or MyHC (1:1000) (Minneapolis, MN, USA) and the secondary antibody IgG (Beyotime, Shanghai, China). Eventually, we measured the enhanced chemiluminescence signal (ECL) (Solarbio, Beijing, China) after adding horseradish peroxidase (HRP) (Bio-Rad, CA, USA) and the GAPDH antibody (1:1000, mouse) (Boster, Wuhan, China) as a loading control.