Western Blot Analysis of Cholesterol Transporters

RAW264.7 cells were cultured on six-well plates with 10% FBS DMEM at a density of 1.0 × 10⁶ cells/well. When the RAW264.7 cells adhered, they were incubated with or without indicated treatment. After the cells were collected, they were washed twice with ice-cold phosphate-buffered saline (PBS). RIPA lysate and protease inhibitor 100:1 were used to extract the protein, and total protein concentration was calculated using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA). The extracted proteins were separated on an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel at 100 V, and the protein was transferred from the gel to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 250 mA for 150 min. The blots were then blocked for 1.5 h at room temperature with skimmed milk powder in 5% bovine serum albumin (BSA). The blots were incubated overnight shaker at 4 °C with mouse anti-ABCA1 (1:500, Abcam, Cambridge, MA, USA), rabbit anti-ABCG1 (1:500, Proteintech, Wuhan, China), rabbit anti-SR-BI (1:1000, Sangon Biotech, Shanghai, China), and rabbit anti-GAPDH (1:1000, Sangon Biotech, Shanghai, China). After rinsing three times for 30 min with TBST, the blots were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:1000, Beyotime, Shanghai, China) or HRP-conjugated anti-mouse IgG (1:1000, CST, USA) for 70 min at room temperature. Lastly, the protein expression was detected using a chemiluminescence Western blotting system.