EMSA of nuclear proteins from HUVECs treated with or without oxidized lipids with the DR4 and Sp1 sequences from the TM promoter. Ox-PAPC and ox-LDL were prepared by UV irradiation of PAPC and LDL, respectively, for 12 hours. Oxidized phospholipids (ox-PLs) and native phospholipids (n-PLs) were separated from crude lipid extracts prepared from their respective LDLs. Reduced forms of ox-PLs (Reduced ox-PLs) and ox-PAPC (Reduced ox-PAPC) were prepared by incubation of the ox-PLs and ox-PAPC with sodium borohydride as described in “Materials and methods.” (A-B) HUVECs were incubated for 9 hours with or without 50 μg/mL ox-PAPC, reduced ox-PAPC, or n-PAPC. (C-D) HUVECs were incubated for 9 hours with or without 200 μg/mL ox-PLs, reduced ox-PLs, or n-PLs. Nuclear proteins (2 μg) isolated from the cells were incubated with 0.5 ng 32P-labeled oligonucleotide probes (1 × 105 cpm) for DR4 (A,C) or Sp1 (B,D) for 30 minutes at 25°C. EMSAs were performed using 4% polyacrylamide gels and complexes bound with oligonucleotide were detected with a Bio-Imaging Analyzer.
