Supershift assay of nuclear proteins extracted from HUVECs with the DR4 sequence and Sp1 binding sequence in the TM promoter. Nuclear proteins were prepared from cultured control HUVECs as described in “Materials and methods.” Nuclear proteins (5 μg) were incubated with or without 1 μg antibody directed against human RARα, RARβ, RARγ, or RXRα protein (A) and Sp1, Sp3, or Sp4 proteins (B) for 30 minutes at 25°C. After incubation, 0.5 ng 32P-labeled DR4 or Sp1 oligonucleotide (1 × 105 cpm) was added, and the reaction was allowed to proceed for 30 minutes at 25°C before EMSA was performed. EMSAs were performed using 4% polyacrylamide gels, and complexes bound with the oligonucleotide were detected with a Bio-Imaging Analyzer (Fuji Film, Tokyo, Japan). Binding specificity of nuclear proteins with the labeled oligonucleotides was confirmed by addition of excess cold DR4 and a scrambled DR4 (A, lanes 6-7) or excess cold Sp1 oligonucleotide (B, lane 2). NS indicates nonspecific binding.
