Western Blot Analysis of Lipid Metabolism Proteins

Protein extraction from cultured cells was performed as described previously [21 (link)]. Samples (30 µg protein/sample) were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Merck). Membranes were blocked for 1 h at room temperature with 5% BSA and incubated overnight at 4 °C with primary antibodies against HMGCR (rabbit anti-HMGCR; ab174830; 1:1,000; Abcam), ACAT-1 (mouse anti-ACAT-1; sc-69,836; 1:1,000; Santa Cruz Biotechnology), LDLR (rabbit anti‐LDLR; ab52818, 1:500; Abcam), and 14-3-3 (rabbit anti-14-3-3, #8312S, 1:1000, Cell Signaling Technology). Membranes were washed, incubated for 1 h with the appropriate secondary antibodies at room temperature, and the immunoblots were developed using enhanced chemiluminescence reagent (ECL plus; Merck). Bands were visualized using the ChemiDoc System. Densitometry of the bands was performed by ImageLab 5.2 version software (Bio-Rad Laboratories, Hercules).