Fig 1: HDLHealthy, but not HDLCAD, decreased LTB4 production from macrophages through proteasome-mediated degradation of 5-LO. Macrophages (RAW 264.7 cell line, 1 × 106) were activated by ZyA (30 min at 37 °C), then treated with HDLHealthy or HDLCAD (10 µg protein). (a) LTB4 production from macrophages was quantified by LC/MS/MS. The results are expressed as pg/1 × 106 cells, mean ± SEM, N = 4-5 in each group. (b) Representative MRM-chromatograph and MS-MS spectrum are presented for the identification of LTB4. (c) Macrophage lysates were processed for western blot analysis of 5-LO. The results are shown as fold changes from N = 4 experiments. (d) 5-LO degradation by HDLHealthy is proteasome-dependent. ZyA-activated macrophages were pretreated for 30 min with the proteasome inhibitor MG 132 (1 µM) or vehicle, then incubated with HDLHealthy for 30 min at 37 °C. Lysates were collected for western blot analysis of 5-LO. The results are shown as the mean ± SEM from N = 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.005. (e) HDLHealthy enhanced ubiquitination of 5-LO in macrophages. ZyA-activated macrophages were pretreated for 30 min with the proteasome inhibitor MG 132 (1 µM) or vehicle, then incubated with HDLHealthy or HDLCAD for 30 min at 37 °C. Lysates were immunoprecipitated with anti-5LO antibody (#3289 S, Cell Signaling), using a Dynabeads Protein A IP Kit (Thermo Fisher Scientific), and then immunoblotted with anti-ubiquitin antibody (ab140601, Abcam). (f) HDLHealthy enhanced 12/15-LO expression in macrophages. Macrophages (RAW 264.7 cell line, 1 × 106) were activated by ZyA (30 min at 37 °C), then treated with HDLHealthy or HDLCAD (10 µg protein, 30 min at 37 °C). After incubation, total RNA was extracted, and then, 12/15-LO expression was analysed by real-time PCR. The results are shown as fold change compared with the vehicle group; mean ± SEM, N = 4. *P < 0.05 vs the other groups.