Physical interaction between RAR/RXR and Sp1. (A) After the mixture of Sp1 plus either RAR or RXR was incubated, respectively, with anti-RAR or RXR antibody at 4°C for 2 hours, proteins were precipitated with protein A-Sepharose, eluted, and analyzed by Western blotting with anti-Sp1 antibody. Lanes 1 through 3, pure proteins; lane 1, Sp1; lane 2, RAR; lane 3, RXR. Lanes 4 through 7, proteins precipitated with anti-RAR or RXR antibody; lane 4, Sp1 preincubated together with RAR and precipitated with anti-RAR antibody; lane 5, Sp1 preincubated together with RXR and precipitated with anti-RXR antibody; lane 6, Sp1 incubated with anti-RAR antibody without RAR; lane 7, Sp1 incubated with anti-RXR antibody without RXR. (B) ³⁵S-labeled Sp1 protein generated by in vitro translation reaction was incubated with GST or RAR-GST immobilized on glutathione-Sepharose beads. After extensive washings, the bound proteins were eluted and subjected to SDS-PAGE followed by autoradiography. Lane 1 input (0.5 μL of the total reaction mixture); lane 2, proteins bound to GST; lane 3, proteins bound to RAR-GST. (C) After the mixture of Sp1 and RAR/RXR was incubated with nonimmune antibody or anti-RXR antibody in BAEC nuclear extracts at 4°C overnight, proteins were precipitated with protein A-Sepharose, eluted, and analyzed by Western blotting with anti-Sp1 antibody. Lane 1, pure Sp1; lane 2, proteins precipitated with nonimmune (NI) antibody; lane 3, proteins precipitated with anti-RXR antibody. (D) BAECs were cotransfected with a combination of Sp1-Gal4 expressing vector (0.125 μg/dish) and Gal4-UAS-luciferase (0.5 μg/dish), plus RAR and/or RXR expressing vectors (250 ng each/dish), along with pRL-CMV (100 ng/dish; Promega) as described before. The day after transfection, the cells were treated or untreated with 1 μmol/L atRA or 9cRA for 12 hours. Luciferase activity of each cell was measured, and relative changes in firefly luciferase activity were plotted after normalization to Renilla luciferase activity. Sample 1, reporter only; sample 2, atRA; sample 3, RAR; sample 4, RAR + atRA; sample 5, RXR; sample 6, RXR + 9cRA; sample 7, RAR and RXR; sample 8, RAR and RXR + atRA; sample 9, RAR and RXR + 9cRA. Each number represents the average ± SD (n = 3). A similar experiment was repeated three times and representative results are shown here.