Lox 1 antibody

Human aortic endothelial cells (HAEC), obtained from Gibco (France), were cultured with M200 medium (Gibco) supplemented with 10% fetal bovine serum (Invitrogen, France) in a humidified atmosphere at 5% CO₂ and 37°C. The cells were used for the experiments after 4 to 5 passages. For the time course of 24 h, OxLDL’s effects on LOX-1 and TGF β protein expressions were evaluated in HAECs treated with culture medium supplemented or not with OxLDL (25 μg/mL) [28 (link),29 ]. We also evaluated TGFβ secretion in culture medium with a Duoset Elisa kit from R&D System (France). The effect of LOX-1 antibody (R&D System), selected for its ability to block receptor-ligand interaction, was tested in these different experimental conditions.

RPMI-1640 culture solution, foetal calf serum (Gibco, Gaithersburg, MD, USA), Ox-LDL, HMCs (Basic Institute of Peking Union Medical College, Beijing, China), IL-1β, LOX-1 antibody (R&D, Abingdon, UK), and Dil-Ox-LDL (Biomed Technologies, Mt. Arlington, NJ, USA) were used. A polymerase chain reaction (PCR) system, reverse transcriptase buffer (Promega, Madison, WI, USA), dNTPs, and Oligo(dT) primers (Sangon Biotech, Shanghai, China) were used. The primer sequences were as follows: LOX‑1 forward, 5'-ACAGAGGCCATTCCGAAATCA-3'; reverse, 5'-GGTAGAGTCTGGAGATGGACCACA-3'; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′; reverse, 5′-ATGGTGGTGAAGACGCCAGT -3′.

H4-II-E-C3 cells (70–80% confluent) were plated in a 6-well culture dish with Dulbecco’s Modified Eagle’s Medium (DMEM; Lonza, Walkersville, MD, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA). Then, 0, 5, 25, 50, 100, 150, and 200 viral particles per cell for 6 h were added. The culture medium was then changed for DMEM 5% and incubated for 48 h at 37 °C at 5% CO₂. Cells were lysed, and western blot followed the previously described protocol using the Lox-1 antibody (R&D Systems, Minneapolis, MN, USA).