Regulation of Cholesterol Metabolism by BB and EG

After being treated with ox-LDL and co-incubated with or without BB (50, 25, 12.5 μmol/mL) and EG (25, 12.5, 6.25 μmol/mL) for 24 h, cellular proteins were extracted with RIPA solution for 30 min and the protein concentrations were then quantified by a BCA protein assay kit (Beyotime). Equal cellular proteins were firstly separated by 8% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes in a pre-cooled transfer solution. After blocking with 5% bovine serum protein for 1.5 h, membranes were incubated overnight at 4 °C with the following primary antibodies: ABCG1 and SR-B1 (Abcam, Cambridge, UK), p-P38, P38, p-P65, P65, GAPDH, β-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA), PPARγ, SR-A1, CD36, P-ERK1/2 ERK1/2, P-JNK, JNK, TLR4 (Affinity Biosciences, Zhenjiang, China) at 1:1000 dilution. Then, membranes were incubated with HRP-conjugated secondary antibody (New England Biolabs, Ipswich, MA, USA) at 1:5000 dilution for 1 h. Finally, enhanced chemiluminescence (ECL) solutions were used to enhance the signal of specific proteins [22 (link)].