1 14c lauric acid

Sources of supplies were: trypticase peptone, tryptone, peptone, glucose, yeast extract, and beef extract, BD Medical Technologies (Franklin Lakes, NJ); haemin, arginine, cysteine, DMSO, CoA and Tween 80, Naja mossambica mossambica snake venom phospholipase A₂, monoclonal anti-polyhistidine-alkaline phosphatase antibody, Sigma-Aldrich (St. Louis, MO); vitamin K1 and media salts, Fisher Scientific (Hampton, NH); Brij-58, G Biosciences (St. Louis, MO); antibiotics, GoldBio (St. Louis, MO); [1-¹⁴C]acetic acid (50.5 mCi/mmol, 1 mCi/ml) and [1-¹⁴C]oleic acid (59 mCi/mmol, 0.1 mCi/ml), PerkinElmer (Waltham, MA); [1-¹⁴C]lauric acid (59 mCi/mmol, 0.1 mCi/ml), American Radiolabeled Chemicals, Inc (St. Louis, MO); (methyl-d₃)12:0, (methyl-d₃)14:0, (7,7,8,8-d₄)16:0, and (methyl-d₃)18:0, Cambridge Isotope Laboratories, Inc (Tewksbury, MA); and methyl ester standards, 14:1(Δ9), 16:1(Δ9), 18:1(Δ9), 18:2(Δ9,12), 18:3(Δ9,12,15), 20:4(Δ5,8,11,14), Matreya, LLC (State College, PA). All solvents were chromatography grade. E. coli ACP was prepared as described (Yao et al., 2013 (link)).

The rabbit P450 4B1 plasmid (4B1 sequence #1)^{23 (link)} was a generous gift from Prof. Allan Rettie (Univ. Washington, Seattle). [1-¹⁴C]-Lauric acid was obtained from American Radiolabeled Chemicals (St. Louis, MO), nickel-nitrilotriacetic acid (Ni-NTA) agarose was purchased from Qiagen (Redwood City, CA), hydroxylapatite agarose was obtained from Bio-Rad (Hercules, CA), and L-α-dilauroyl-sn-glycero-3-phospocholine (DLPC) was obtained from Enzo Life Sciences (Farmingdale, NY). All other reagents were obtained from either Sigma Aldrich or Fisher Scientific.

Anti-human CYP2J2 antibodies used for immunoblot analysis were prepared as described previously (King et al. 2002 (link)). Anti-rat CYP4A antibodies and anti-human CYP4F11 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-human CYP4F3 antibodies were purchased from Abnova (Taipei, Taiwan). Anti-human CYP4F12 antibodies were purchased from Abcam (Cambridge, MA). The secondary antibodies (goat anti-mouse, donkey anti-goat, and goat anti-rabbit horseradish peroxidase-conjugated IgGs) were purchased from Santa Cruz Biotechnology. Polyvinylidene difluoride (PVDF) membranes and reagents for enhanced chemiluminescence were purchased from GE Healthcare (Piscataway, NJ). [1-¹⁴C] Lauric acid was purchased from American Radiolabeled Chemicals (St. Louis, MO). All other chemicals and reagents were of electrophoresis or analytical grade where appropriate.