Fig. 4 |. AKBA modulates the enzymatic activity of purified human 5-LOX in a cell-free assay.

Purified human recombinant 5-LOX (0.5 μg ml⁻¹ PBS pH 7.4 plus 1 mM EDTA) was preincubated with increasing concentrations of AKBA (blue), NDGA (white) or with vehicle (0.1% DMSO, black) on ice for 15 min. The samples were incubated with 2 mM CaCl₂ and 10 μM AA at 37 °C. a-d, After 10 min, the reaction was stopped on ice and formed 5-LOX products (lipid mediators (LM) 5-HETE (a); LTB4 isomers (b); 12-HETE (c) and 15-HETE (d)) were analyzed by UPLC-MS/MS. Data obtained in n = 3 independent experiments were log-transformed for statistical analysis; *P < 0.05 AKBA versus vehicle control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 NDGA versus vehicle control; ANOVA and Dunnett’s multiple comparisons test.