Oleoyl coa

2.8 mM cholesterol/11.2 mM PC/18.6 mM taurocholate were mixed to generate the cholesterol donor^{47 (link)}. The tetrameric or dimeric hSOAT1 enzyme was prepared in GDN detergent. First, 10 μl 2 M KCl, 5 μl 5% BSA, 1 μl SOAT1 protein (A₂₈₀ = 0.5), 5 μl micelles, 40 μl TBS buffer with 40 μM GDN were mixed with TBS buffer with 0.5% CHAPS to reach the volume of 100 μl and incubated at 37 °C for 2 min. In order to measure the IC₅₀ of inhibitors, different concentrations of given inhibitors were added, as indicated. Then, 1.0 μl 0.2 mg ml⁻¹ NBD-cholesterol (Sigma, N2161) solubilized in 35% β-cyclodextrin (Sigma, HZB1102) was added and the mixture was incubated at 37 °C for 2 min. To start the enzymatic reaction, 1 μl 2.5 mM oleoyl-CoA (Sigma, O1012) was added and the reaction mixture was incubated at 37 °C for 15 min. The reaction was terminated by adding 2:1 chloroform/methanol, the extract was separated on an HPLC column at 0.2 ml/min (Agilent, Poroshell HPH-C18, 2.7 μm) running in 100% ethanol and detected via fluorescence detector on an HPLC (SHIMADZU). NBD-cholesterol eluted at 1.3 min and its ester eluted at 1.9 min. The peak areas of the NBD-cholesteryl ester products and remaining NBD-cholesterol were integrated separately to obtain the relative ratio of NBD-cholesterol that was converted into NBD-cholesteryl esters.