DNA-bound RARβ interacts with Sp1/Sp3 following RA treatment. (A) EMSA was performed following incubation of the wild-type GC-box probe with 2.5 μg nuclear proteins extracted from HUVECs that had been treated with 10^–6 M RA for various time periods (0, 4, 6, 8, or 24 hours). The slight increase in complex intensity at 6 hours (A, lane 3) indicates that RA treatment enhances the binding of Sp1 and Sp3 to the t-PA –7351 site. (B-C) Nuclear proteins (15 μg) from nontreated HUVECs (B) and 6-hour RA-treated (10^–6 M) HUVECs (5 μg; C) were incubated with a ³²P-labeled DR5 oligonucleotide, with or without the addition of antibodies directed against human RARα, RARβ,orRARγ as indicated. Binding specificity of nuclear proteins with the labeled oligonucleotide was confirmed by addition of excess cold DR5-specific competitor (B and C, lane 2) and nonspecific competitor (B and C, lane 3). NS indicates nonspecific. The results show that RARγ binds to the DR5 site and that RARβ is present at the DR5 site during RA-treated conditions. (D) Nuclear proteins (5 μg) from 6-hour RA-treated (10^–6 M) HUVECs were incubated with a ³²P-labeled GC-box/DR5 oligonucleotide, with or without the addition of antibodies directed against human Sp1, Sp3, Sp4, RARα, RARβ,orRARγ as indicated. Supershifting was performed as described in the “Materials and methods,” except that the RAR antibodies were added prior to that of the labeled oligomer. Lanes 1 and 5 represent control lanes with no added antibodies. Addition of RARβ antibodies (D, lane 7) inhibits formation of the Sp1 and Sp3 containing complexes (complex A, B, and C), suggesting that RARβ is present in combination with Sp1 and Sp3 in these complexes.