Potentiation of Sp1 binding to uPA GC box by RAR and/or RXR. (A) After Sp1 (10 to 20 ng) was preincubated in the absence or presence of RAR-GST (200 ng), the reaction mixture was incubated with ³²P-labeled uPA GC box or consensus GC box, and thereafter protein-DNA complexes were separated by a 4% polyacrylamide gel electrophoresis and visualized on an image analyzer. Odd numbers, Sp1 alone; even numbers, Sp1 plus RAR. (B) Gel shift analyses were performed as before using both RAR-GST and RXR-GST. Lane 1, Sp1 (10 ng) alone; lane 2, GST alone; lane 3, Sp1 plus GST; lane 4, RAR alone; lane 5, Sp1 plus RAR; lane 6, RXR alone; lane 7, Sp1 plus RXR; lane 8, RAR and RXR; lane 9, Sp1 plus RAR/RXR. (C) Control experiments. Lane 1, control (Sp1 [10 ng] plus RAR/RXR); lane 2, + 20-fold excess of unlabeled oligonucleotide (cold); lane 3, + 50-fold excess of unlabeled oligonucleotide (cold); lane 4, + 0.5% ethanol (vehicle); lane 5, + 1 μmol/L atRA; lane 6, + 1 μmol/L 9cRA; lane 7, + nonimmune (NI) antibody (IgG); lane 8, + anti-Sp1 IgG; lane 9, + anti-RAR IgG; lane 10, + anti-RXR IgG; lane 11, + both anti-RAR IgG and anti-RXR IgG. The final concentration of each antibody was 80 μg/mL.