Nefa kit

The coding sequences from BnaC.MAGL8.a and AtMAGL8 were inserted into the pMAL-c5X vector from the MBP purification system kit (NEB). After confirmed by sequencing, the vectors were introduced into E. coli strain ER2523. Protein purification and western blot were performed according to the manufacturer’s instructions. After dialysis against 50 mM phosphate sodium solution (pH 8.0), the maltose binding protein (MBP):MAGL recombinant proteins were quantified using Bradford protein assay kit (Tiangen). The recombinant proteins were incubated with the substrates (Supplementary Table S2), MAGs with various fatty acids, including palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2), at sn-1 position and the MAG with 18:1 at sn-2 position in 100 μl reaction solution containing 50 mM sodium phosphate buffer (pH 8.0), 0.2% Triton X-100 (Kim et al., 2016 (link)). All substrates were bought from Sigma-Aldrich and were emulsified in 0.2% Triton X-100 solution before use. After 30 min incubation, the reaction was stopped by 90°C treatment for 5 min and the released free fatty acid was measured by NEFA kit (Wako).

FFA concentration was measured using 2 μl serum with the NEFA kit (WAKO). Briefly, 75 μl Reagent A was mixed with the serum, and incubated at 37°C for 5 minutes and then absorbance was measured at 550 nm (reference 660 nm) using the manufacturer’s protocol. The 150 μl Reagent B was added, and incubated at 37°C for 5 minutes, then absorbance was measured again. A standard curve was us to determine sample concentrations.