Protein assay kit

L6 myoblasts were cultured at 90% confluence and differentiated. Myoblasts and differentiated myotubes were homogenized with lysis buffer (25 mM Tris, pH 7.9, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 5% glycereol, protease inhibitors). Protein concentration of cell lysates was determined by the Protein Assay Kit (iNtRON biotechnology, Korea), an equal amount of cells lysates was separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Germany). The membranes were incubated with specific antibodies against MHC, p-AKT, AKT, p-ERK, ERK, and GAPDH. The blots were then incubated with horseradish peroxidase conjugated anti-mouse secondary antibodies or horseradish peroxidase conjugated anti-rabbit secondary antibodies. Bands were detected by the ECL detection system (GE Healthcare, Germany) and band intensities were quantified by ImageJ software (Yi, Hwang, Oh, Kim, Ryu, et al. 2018 (link)).

For measurement of the osteogenic activity of cells on the scaffolds, alkaline phosphatase (ALP) activity and mineralization assays were performed. Cells were seeded at a density of 1 x 10⁴ and cultured in osteogenic media (α‐MEM with 15% FBS supplemented with 0.2 mM ascorbic acid [Gibco] and 10 mM β‐glycerol phosphate [Gibco]). On days 7 and 14 of culture, ALP activity was measured using an ALP Activity Assay Kit (AnaSpec Inc.,Fremont., CA) and a Protein Assay Kit (iNtRON Biotechnology, Seoul, Korea). ALP activity was normalized as the concentration of p‐nitrophenol (pNP) to the protein amounts. On the day 21, mineralization analysis was performed. Fixation with 95% cold ethanol was done. Cells were stained with a 1% alizarin red S solution (Wako Chemicals, Osaka, Japan) for 5 min.
Cells were fixed in 95% cold ethanol and stained with the images of stained plates were observed. For quantitative measurement, an elution procedure with cetylpyridinium chloride in 10 mM sodium phosphate was performed and measured using an automatic microplate reader at 540 nm wavelength.

Whole cell lysates were prepared with lysis buffer (25 mM Tris, pH 7.9, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 5% glycerol, protease inhibitor cocktails, 1 mM sodium orthovanadate, and 2.5 mM sodium pyrophosphate). Protein concentration of cell lysates was determined by the Protein Assay Kit (iNtRON biotechnology, Inc., Seoul, Korea). Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Freiburg, Germany), and subject to Western blotting using the indicated antibodies. The antibodies used in this study were as follows: p-Akt, Akt, p-ERK and ERK antibodies from Cell Signaling Technology (Danvers, MA, USA); p-IκB, NFATc1 and p65 antibodies from Santa Cruz (Santa Cruz, CA, USA); IκB and β-Actin antibodies from Sino Biological (Beijing, China); and p-p38, p38, p-JNK, JNK, and c-Fos antibodies from Millipore (Burlington, MA, USA).