1 14c oleoyl coa

Erythrocyte LPCAT activity was determined by measuring the formation of [¹⁴C] PC from lysoPC and [¹⁴C] acyl-CoA26 (link). All reactions were performed in 100 mM Tris-HCl, pH 7.4 containing 1 μM CaCl2, 0.015% Tween-20, 200 μM lysoPC (Avanti Polar Lipids), and 20 μM [1-¹⁴C] oleoyl-CoA (0.01 μCi) (Perkin Elmer) at 37 °C with isolated erythrocyte membrane protein in a total volume of 200 μl. The reaction was terminated by adding 800 μl of chloroform/methanol (2:1, vol/vol) to the incubation mixture. Then lipids were extracted and then resolved by Thin-layer chromatograph (TLC) silica plates with chloroform/methanol/acetic acid/0.9% NaCl (100:50:16:5, vol/vol). TLC plates were then exposed to phosphor-imaging screening (Bio-Rad) and scanned for radioactive signals as indications of the formation of PC. A part of membrane protein was used to detect LPCAT1 protein expression by Western blot. Approximately 20 μg membrane protein was run on 10% SDS-PAGE gels. After protein was transferred to PVDF membrane, the membrane was blocked with 5% nonfat milk and incubated with anti-LPCAT1 antibody (ab94903, Abcam) and Donkey anti-Rabbit secondary antibody (Santa Cruz Biotechnology Inc.), respectively.

A TAG assay kit was purchased from Cayman Chemicals (Ann Arbor, MI). The ApoB ELISA kit was from LifeSpan Bioscience (Nottingham, UK). The RNeasy Mini kit was bought from Qiagen (Manchester, UK). [1-¹⁴C]-oleoyl-CoA (specific activity 60 mCi/mmol) was purchased from PerkinElmer (Coventry, UK). A cholesterol assay kit, Triton WR1339, tetrahydrolipstatin (THL), DGAT1 inhibitor T863, and all primers were purchased from Sigma-Aldrich (Irvine, UK). DGAT2 inhibitor (iJ) was prepared by Tocris Bioscience (Abingdon, UK). Heparinized syringes were from Abaxis UK Ltd. (York, UK).

BeauI and BeauIII were purified from the culture broth of the producing fungus Beauveria sp. FO-6979 according to our established methods (Supplementary Fig. 3)^{1 (link)}. Biotin-labeled beauveriolide (Biotin-Beau) was synthesized as reported previously (Supplementary Fig. 3)^{11 (link)}. [1-¹⁴C]Oleic acid (2.19 GBq/mmol) and [1-¹⁴C]oleoyl-CoA (1.85 GBq/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). 2.5% Trypsin and acrylamide-based streptavidin beads (Streptavidin UltraLink Resin) were purchased from Thermo Fisher Scientific (Waltham, MA). Triton X-100 was purchased from Sigma-Aldrich (St. Louis, MO). Digitonin were purchased from Wako Pure Chemical Industries (Tokyo, Japan). Saponin was purchased from MP Biomedicals (Santa Ana, CA). OptiPrep^TM was purchased from Axis-Shield (Luna Place, Scotland). An anti-rabbit IgG conjugated to horseradish peroxidase (HRP) was purchased from Medical & Biological Laboratories (Nagoya, Japan).