Bca protein quantification kit

The HepG2 cell line, the Caco-2 cell line, filipin, Dulbecco’s modified Eagle’s medium, penicillin/streptomycin (batch number PWL062), and the BCA protein quantification kit (batch number MA0082-2) were purchased from Meilun Bio., Dalian, China; fetal bovine serum (FBS) was purchased from Gibco, Billings, MT, USA; dimethyl sulfoxide (DMSO, lot number D8371) and the cell viability CCK-8 kit (lot number MA0718-L) were purchased from the Biyuntian Institute of Biotechnology, Shanghai, China. NBD-cholesterol was purchased from Bailingwei, Beijing, China; PTL, mevalonate, and methyl-β-cyclodextrin (CDX) were purchased from Sigma, New York, NY, USA; lovastatin and cholesterol were purchased from Macklin, Shanghai, China; defatted fetal bovine serum (LPDS) was purchased from Biological Industries, Israel.

RIPA buffer (Solarbio, China) supplemented with protease inhibitor (Roche, Germany) was used to homogenize and lyse muscle tissue samples to obtain total protein extracts. Protein concentration of protein extracts was measured using a BCA Protein Quantification Kit (Meilunbio, China) according to the manufacturer’s guidelines. The total of 30 µg protein of each sample was run on 10% SDS-PAGE gels in electrophoresis buffer and transferred to nitrocellulose membranes (Millipore, USA) for blotting. Immunoblots were blocked for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies with the following antibodies: rabbit anti-MyoD1 (1:1000, Wanleibio, China) and mouse anti-tubulin (1: 1000, Poteintech, USA). Goat anti-rabbit IgG-HRP (CWBIO, China) and goat anti-mouse IgG-HRP (Beyotime, China) were used as secondary antibodies and protein signals were detected using enhanced chemiluminescence (ECL) (Millipore, USA).